Outline of Blood and Body Fluid

Body Fluid
1.0 Introduction
1.1 Significance of body fluid
1.2 Distribution of body fluid/ composition
1.3 Cerebrospinal fluid
1.4 Seminal fluid
1.5 Synovial fluid
1.6 Amniotic fluid
1.7 Intra-ocular fluid
1.8 Sweat/ urine
1.9 Saliva/ Human milk
Blood
1.0 Introduction
1.1 Properties of blood/ composition
1.2 Functions of blood
1.3 Plasma Proteins
1.3.1 Separation of plasma proteins
1.3.2 Albumin
1.3.3 Globulin
1.3.4 Fibrinogen
1.3.5 Functions of plasma proteins
1.4 Haemoglobin
1.5 Blood coagulation
1.6 References

BLOOD AND BODY FLUIDS

Introduction: The body is formed by solids and liquids. The fluid part is more than
2/3 of the whole body and is mostly water. In human beings, the total body water
varies from 45 to 75% of body weight. In normal young adult male, body contains
60-65 % of water and 35 - 40% of solids. In a normal young adult female, the
water is 50 to 55% and solid are 45 - 50%. In females, water is less because of
more amount of Subcutaneous adipose tissue. The total quantity of body water in
an average human being weighing about 70kg is about 40 litres.

SIGNIFICANCE OF BODY FLUIDS:

1. In Homeostasis: Body cells survive in the fluid medium called Internal

environment. Water forms not only the essential constituent of internal
environment but also plays an important role in homeostasis. Growth and
functions of cells also depend upon the availability of certain materials like
glucose, amino acids, lipids, vitamins, ions, oxygen etc in proper quantities in
the internal environment.
2. In Transport Mechanism: Body water forms the transport medium by which
nutrients and other essential substances enter the cells, and unwanted
substances leave the cell. Also water forms an important medium by which
various enzymes, hormones, vitamins, electrolytes and other substances are
carried from one part to another part of the body.
3. In Metabolic Reactions: Metabolic reactions, which are necessary for growth
and functional activities of the cells occur via water inside the cells.
4. Texture of Tissue: Water inside the cell is necessary for the characteristic form
and texture of various tissues.
5. In Temperature Regulation: Water plays a vital role in the maintenance of
normal body temperature.

COMPACTMENTS/ DISTRIBUTION OF BODY FLUIDS

Plasma Total water in the body is about 40litres. It is

distributed into two major fluid compartments:

1. Intracellular Fluid (ICF) forming 55% of the total body water (22litres)

2. Extracellular Fluid (ECF) forming 45% of the total body water (18litres)

ECF is divided into 5 subunits:

I. Interstitial fluid and lymph

II. Plasma

III. Fluid in bones

IV. Fluid in dense connective tissues like cartilage and

V. Transcellular fluid that includes:

a. Cerebrospinal fluid

b. Intraocular fluid
c. Digestive juices

d. Serous fluid - intrapleural fluid, pericardial fluid, and peritoneal fluid.

e. Synovial fluid in joints

f. Fluid in urinary tract.

The volume of interstitial fluid is about 12litres. The volume of plasma is about
2.75litres; others 3.25litres. NOTE - water moves between different
compartments.

(2.75L)
Extracellular fluid

Interstitial Intracellular fluid

(18L)

Fluid (22L)
(12L)

Other fluid
(3.25L)

COMPOSITION OF BODY FLUIDS

Body fluids contain water and solids. Solids are organic and inorganic substances.
Organic substances - are glucose, amino acids and protein, fatty acids and other
lipids, hormones and enzymes.

Inorganic substances - Those present in body fluids are sodium, potassium,

calcium, magnesium, chloride, bicarbonate, phosphate and sulfate.

Extracellular fluid contains large quantity of sodium, chlorides, bicarbonate,

glucose, fatty acids and oxygen. Intracellular Fluids contains large quantities of
potassium, magnesium, phosphates, sulfates and proteins. The PH of Extracellular
Fluid is 7.4, the PH of Intracellular Fluid is 7.0.

CEREBROSPINAL FLUID (CSF)

Definition: The fluid contained in the central canal of the spinal cord,
subarachnoid space and the cerebral ventricles is known as CSF. It is a part of ECF.

Normal amount is about 150ml (range 100 - 200ml)

CFS has various constituents which include; cells, proteins, anions, glucose etc. It
has lower concentration of non-diffusible anions like proteins than plasma but has
higher concentrations of some anions like chloride than plasma. Most electrolytes
in the other body fluids are higher than plasma.

SITE OF FORMATION

CSF is formed by the choroid plexuses situated within the ventricles. The choroid
plexuses are tuft or bed of capillary projections present inside the ventricles and
are covered by pia mater and ependymal covering. (note: A large amount of CSF
in formed in lateral Ventricle)
Mechanism of formation: CSF in formed by the process of secretion. It does not
involve ultra filtration or dialysis but active transport mechanism is involved in the
secretion as energy is expended

FACTORS AFFECTING THE FORMATION OF CSF

1. Pilocarpine, ether and extracts of pituitary gland stimulate the secretion of

CSF by stimulating the choroid plexuses.

2. Isotonic Saline injection also stimulates CSF formation.

3. Injection of hypotonic saline causes greater rise in capillary pressure and

intra-cranial pressure and fall in osmotic pressure leading to increase in CSF
formation.

4. Hypertonic saline decrease CSF formation and decrease the CSF pressure.
(The increased intracranial pressure is decreased by injection of 30 - 35% of
NaCl or 50% sucrose).

PROPERTIES OF CSF

1. Color - CSF is a clear, colorless, transparent fluid.

2. Volume - 150ml

3. Rate of formation - 0.3ml per minute

4. Specific gravity - 1.005 - 1.006

5. Reaction - Alkaline
 COMPOSITION OF CSF

- Consist of water - 99.13% and solids 0.87%

The solid consists of organic and inorganic substances.

Organic substances present include - proteins, amino acids, sugar, cholesterol,

urea, Uric acid, creatinine and lactic acid.

The inorganic substances are – Sodium, Calcium, Potassium, Magnesium,

Chloride, phosphate, Bicarbonate and sulfates.

CSF contains more sodium than potassium. It also contains lymphocytes which are
added when it flows in the spinal cord. The CSF Secreted by the ventricle does not
contain any cell.

CIRCULATION OF CSF

Large quantity of CSF is formed in the lateral ventricles and passes through the
foramen of Monro into the 3rd ventricle. From here, it passes into the 4th
ventricle Via aqueduct of Sylvius. From 4th Ventricle, it inters into the Cisterna
magna and Cisterna lateralis through the foramen of Magendie (central opening)
and foramen of Luschika (lateral opening). The greater portion of the fluid passes
upwards, over the brainstem to the surface of the cerebral hemispheres while a
portion of the cisternal fluid circulates through the spinal subarachnoid space
 Superior sagittal venous sinus

Dura mater

Arachnoid mater

Subarachnoid space

Pia mater

Choroid plexus
Foramen of Luschika Lateral Ventricle
Third Ventricle
Foramen of Magendie Aqueduct of sylvius
Fourth ventricle
Choroid plexus
Central Canal of Spinal cord
Dura mater
Arachnoid mater
Pia mater

ABSORPTION: Mostly absorbed by the arachnoid villi into dural sinuses and spinal
veins. Small amount is absorbed (along the perineural spaces) into cervical
lymphatics and into the perivascular spaces.

The mechanism of absorption is by filtration due to pressure gradient between

hydrostatic pressure in the subarachnoid space fluid and the pressure that exists
in the dural sinus blood. Normally about 500ml of CSF is formed every day and an
equal amount is absorbed.

Pressure Exerted by CSF - Exerts a pressure of 10 - 18cm of H 2O in lateral

recumbent position and 30cm of H2O in the sitting position. Certain events like
coughing, and crying increase the pressure by decreasing the absorption.

Also, compression of Internal Jugular Vein increases the CSF pressure.

FUNCTIONS OF CSF

1. Protective Function: It acts as fluid buffer and protects the brain from shock.
The brain floats in CSF (because the sp. gravity of brain and CSF is approximately
the same), thus when head receives a blow, CSF acts as a cushion and prevents
the movement of brain against the skull bone, thereby preventing the damage of
brain.

However, in the event of severe blow, the brain moves forcefully and hits against
the skull bone leading to damage of the brain tissues. The brain strikes against the
skull bone at a point opposite the point of impact (where the blow was applied),
hence called counter coup injury.

2. Medium of Exchange: Serves as medium through which many substances,

especially the nutritive substances and waste materials are exchanged between
blood and brain tissues.

3. Regulation of Cranial Content Volume: Greater absorption of CSF helps in

regulation of cranial content volume. This is essential if the cranial contents
increase in volume e.g in cases of cerebral hemorrhage and tumors in the brain.
When substances are absorbed into the venous sinuses, intracranial pressure is
raised which interferes with the cerebral circulation causing asphyxia.

COLLECTION OF CSF - Collected either by cisternal puncture or lumbar puncture.

In cisternal puncture the CSF is collected by passing a needle between the
occipital bone and atlas, so that it enters the cisterna magna. In lumbar puncture,
the lumber puncture needle is introduced into the subarachnoid space in the
lumbar region, between the 3rd and 4th lumbar spines.

Reasons for selecting site of lumber puncture:

- The spinal cord will not be injured, because it terminates below the lower
border of the 1st lumbar vertebra. The cauda equina may be damaged but it is
regenerated.

- The subarachnoid space is wider in this site because the pia mater is reduced
very much.

Purpose of lumber puncture:

1. For diagnostic purposes

2. For introducing spinal anesthesia

3. For measuring pressure exerted by CSF

HYDROCEPHALUS: The abnormal circulation of CSF in the skull associated with

enlargement of head is called hydrocephalus. It could be Noncommunicating
(Internal) hydrocephalus during which there is obstruction to any of the foramen
through which CSF escapes leading to dilatation of Ventricular cavity. External or
Communicating Hydrocephalus occurs if the arachnoid villi are blocked.
Symptoms - Headache, vomiting. In severe conditions, leads to atrophy of brain,
mental weakness and convulsions.

Composition of CSF

Parameter Findings

1. Color and appearance Clear and colorless

2. Protein content 10 - 30mg/dl

3. Glucose Usually 2/3 of plasma level (50 - 70mg/dl)

4. Cell count 0 - 3 x 106/liter

Clotting (coagulation) Normal CSF does not clot

However, in disease conditions like infections, tumors, CNS bleeding

(hemorrhage) etc., the normal CSF composition as indicated above is altered.
These alterations are valuable pointers to the likely causes of diseases e.g.

i. Bacterial meningitis: the CSF is turbid, has increased protein content, markedly
reduced glucose concentration and increased cell count predominantly in the
form of Neutrophils. The CSF may also clot if left standing for some time.

ii. Tuberculous Meningitis: The cell count is increased but unlike in plain bacterial
meningitis the predominant cells are lymphocytes. The protein content is
increased (usually much higher than in bacterial meningitis), the glucose content
is low (the values however are not as low as in bacterial meningitis), and the fluid
usually will clot on standing with a characteristic cobweb appearance. The fluid is
usually a bit opalescent.
iii. Viral Meningitis: the CSF is colorless and clear, the glucose content is normal
but there is markedly increased protein content and also increased cell count
predominantly the lymphocytes.

iv. Subarachnoid hemorrhage: In this situation the CSF is evenly blood stained
(sometimes deep amber color). There is increased RBCs and WBCs in the cell
count and increased protein content. However, there is usually no change in the
glucose content and no coagulation or clot formation.

SEMINAL FLUID (SEMEN)

It is an organic fluid that usually contain spermatozoa and is secreted by the

gonads and other sexual organs of the male which include: prostate, seminal
vesicle, bulbo urethra gland. In appearance, it’s usually white but Grey /slightly
yellow colored semen may also be normal. It is sterile with PH that varies
between 7.2 - 8.0 and the fluid is usually released as ejaculate with a volume of
greater or equal to 2ml per ejaculate taken as normal. It contains several
components beside the spermatozoa and these include:

1. Proteolytic and other enzymes e.g. PSA, Acid phosphatase, Glycosidase,

Fibrinolysin etc.

2. Monosaccharides e.g. fructose, Galactose

3. Citric acid

4. Lipids e.g. prostaglandins (PG E1 and E2), inositol glyceryl

phosphorylcholine
5. Micronutrients e.g. Zinc, mg, Se (most especially zinc)

6. Others- Mucus, Salicylic acid, Flavins, Carnitine.

Seminal fluid is primarily water and has slightly higher amount of K+, Zn 2+, Mg2+,
Se than plasma

Gland % contribution to Constituents Release

Seminal fluid

Testes 2 - 5% Approximately 200 - 500 million spermatozoa per

ejaculate

Seminal 65 - 75 % Amino acid, citrate, Flavins, prostaglandins,

Vesicle vitamin C, phosphorylcholine

Prostate 25 - 30% Acid phosphatase (ACP), PSA, Citric acid,

Fibrinolysin, zinc, other Proteolytic enzyme.

Bulbourethra <1% Mucous, galactose, fructose, sialic acid etc.

gland

Some functions of seminal fluid constituents

1. Fructose - the main energy source for spermatozoa

2. Prostaglandins - Involved in the suppression of immune response by the

female immune system against the foreign spermatozoa.

3. Zinc - helps to stabilize the DNA containing chromatin in the sperm.

4. Basic amines – e.g. putrescin, spermidine, spermine, cadaverine. They
counteract the acidic environment of the vagina canal and protect the DNA
inside the cell from acidic denaturation.

5. Mucus - Create a reduced viscous channel for the sperm to swim and thus
assist sperm motility. It also contributes to the cohesive jelly-like nature of
the semen.

SYNOVIAL FLUID

Is a yolk-like fluid found in the cavities of synovial joints and functions to reduce
friction between the articular cartilages of synovial joints during movement. It is
secreted by synovial membrane and two types of cells make up this synovial
tissue; type A and type B; it is the type B cell that produce the fluid. This
membrane does not have basement membrane and hence there is no obstruction
to the fluid flow.

The normal constituent of synovial fluid.

1. Hyaluronic acid

2. Lubricin

3. Phagocytic cell

Normal synovial fluid is;

a. Colorless or straw color with a high viscosity.

b. It exhibits non-Newtonian flow characteristics, in that the viscosity

coefficient is not a constant and the fluid is not linearly viscous. A non-
 Newtonian fluid is a fluid that does not follow Newton’s law of viscosity,
that is, it has variable viscosity dependent on stress. In non-Newtonian
fluids, viscosity can change when under force to either more liquid or more
solid. Newton’s viscosity law states that viscosity should remain constant
regardless of stress.

c. It has thixotropic property i.e. Viscosity decreases and the fluid thins over
period of stress or movement.

The Hyaluronic acid component of synovial fluid act to increase the viscosity and
elasticity of articular cartilage, and lubricate the surfaces between the synovium
and cartilage.

Lubricin, on the other hand is involved in the boundary layer lubrication which
reduces friction between the opposing surfaces of cartilage. Lubricin is also
believed to be involved in regulation of cell growth.

Phagocytic cells remove microbes and cellular debris that result from normal
wear and tear in the joint.

In the presence of infection or inflammation, the constitution of synovial fluid

changes and this can be a guide to diagnosis when synovial fluid analysis is done.

Synovial fluid also; assist in shock absorption, transport O 2 and nutrients and
removes CO2 and waste metabolites from the chondrocytes within the articular
cartilage.

Normal Non-Inflammatory Inflammatory Septic

Volume (ml) <3.5 >3.5 >3.5 >3.5

Color Colorless or straw/ yellow yellow mixed
straw colour
Clarity Clear clear cloudy opaque
WBC <200 200 – 2000 200 – 75000 >100,000
Polymorph <25% <25% >50% >75%
AMNIOTIC FLUID

This play a major role in Fetal growth and development and as regard its
production; some amniotic fluid is initially secreted by amniotic cell but most is
derived from maternal tissue and interstitial fluid by diffusion across the Amnio
choric membrane from decidual basalis.

Composition of Amniotic fluid.

Is an aqueous solution in which undissolved material (desquamated fetal

epithelial cells) is suspended. Amniotic fluid contains approximately equal
portions of organic and inorganic constituents. For the organic constituents,
about half is made up of protein and the other by constituents such as
carbohydrates, fats, enzymes, hormones, pigments etc.

Early Gestation Pre-term

Volume 450 – 1200ml 500 – 1400ml
Bilirubin <0.075mg/dl <0.025mg/dl
Creatinine 0.8 – 1.1mg/dl 1.8 – 40mg/dl
Estriol 10ug/dl >60ug/dl
L/s ratio <1.1 >2.1
Protein 0.6 – 0.24g/dl 0.26 – 0.19g/dl
Urea 18 ± 6mg/dl 30 ± 11mg/dl
Uric acid 3.7 ± 1mg/dl 9.9 ± 2.2mg/dl

L/S = Lecithin / sphingomyelin

Steroid administration in pregnancy is to stimulate lung development i.e
surfactant production.

CLINICAL SIGNIFICANCE /APPLICATION OF AMNIOTIC FLUID

Amnioncentesis: Is the process by which amniotic fluid is collected for analysis

and this analysis can be very important in prenatal diagnosis in condition such as:

a. Assessment of lung maturity: The lecithin/sphingomyelin ratio is usually

used to assess lung maturity and most people would accept that a value
greater than 2.0 is a pointer to a mature lung.

b. Assessment of Hemolytic disease: The measurement of Bilirubin in

amniotic fluid is useful for early detection of hemolytic disease of Newborn.

c. Use of alpha-fetoprotein in the detection of neural tube defect (NTD),

down Syndrome (Trisomy 21), Trisomy 18 (Edward syndrome) etc.

d. Alpha-cholinesterase: Estimation of this in amniotic fluid can also be useful

in detection of neural tube defect.

Note: The analysis of most of these biochemical substances are interpreted taken
into cognizance the gestational age i.e. the levels are variable depending on the
duration of the pregnancy.

Lung maturity: Is assessed biochemically by measuring Lecithin and

Sphingomyelin in amniotic fluid and calculating the L/S ratio which is an index of
the surfactant concentration in the fetus. In late pregnancy the cells lining the
fetal alveoli starts synthesizing lecithin (Depamitoylphosphatidalcholine) so that
its level rises whereas that of Sphingomyelin remain unchanged; as a result, the
L/s ratio rises. A ratio of > 2 is indicative of lung maturity.

INTRA-OCULAR FLUID

This is the fluid inside the eye. It is responsible for keeping the eye distended and
maintaining the eye in its spherical form. The fluid can be divided into two parts:
A. The aqueous humor: Is a thick watery substance filling the space between the
lens and the cornea. It is transparent so as to allow light to pass through it.

Functions of Aqueous humor

I. Maintains the Intraocular pressure and inflates the globe of the eye.

II. Provides nutrition (e.g. amino acids and glucose) for the avascular ocular
tissues; posterior cornea, trabecular meshwork, lens and Anterior Vitreous

III. May serve to transport ascorbate in the anterior segment to act as an anti-
oxidant agent.

IV. Presence of immunoglobulin indicates a role in immune response to defend

against pathogens.

V. Provides inflation for expansion of the cornea and thus increased

protection against dust, wind, pollen grains and some pathogen.

VI. For refractive index

Composition of Aqueous humor: Amino acids transported by ciliary epithelial

cells.

Production and Drainage: Aqueous humor is secreted into the posterior chamber
by the ciliary body specifically the non-pigmented epithelium of the ciliary body
(pars plicate). It drains into the Schlemm’s canal by one of two ways and
eventually into the episcleral blood vessels via aqueous veins.

Glaucoma is a condition characterized by increased Intraocular pressure either

through increased production or decreased outflow of aqueous humor.
Uncontrolled glaucoma typically leads to visual field loss and ultimately blindness.
Imbalance between formation and reabsorption of aqueous humor is what
usually lead to a rise in Intraocular pressure (i.e. 20 - 30mmHg) which if persistent
for a long period of time can cause blindness which ensues from the compression
of neural cells of the retina by Glaucoma. Glaucoma may also compress retinal
vessels which worsen and speed up blindness.

B. Vitreous humor: This is the clear gel that fills the space between the lens and
the retina of the eyeball of humans and other vertebrates.

Description of Vitreous humor: It is the transparent, colorless gelatinous mass

that fills the space between the lens of the eye and the retina lining the back of
the eye. It is of rather similar composition to the cornea, but contains very few
cells (mostly phagocytes which remove unwanted cellular debris in the visual field
and the hyalocytes of Balazs of the surface of the vitreous, which reprocess the
hyaluronic acid). It has no blood vessel, 98 - 99% of its volume is water with salts,
sugar, vitrosin (a type of collagen), a network of collagen type II Fiber with the
glycosaminoglycan hyaluronic acid and a wide array of patterns in micro-amounts.
Viscosity is 2 - 4 times that of pure water giving it a gelatinous consistency. Its
refractive index is 1.336.

Clinical significance: The metabolic exchange and equilibration between systemic

circulation and vitreous humor is so slow that vitreous humor is sometimes the
fluid of choice for postmortem analysis for glucose levels or substance which
would be more rapidly diffused, degraded, excreted or metabolized from the
general circulation.

SWEAT

Sweat is a fluid consisting primarily of water as well as various dissolved solids,

chiefly chlorides that is excreted by the sweat glands in the skin of mammals.
Sweat contains the chemicals or odorants 2 - methyl phenol and 4 - methyl
phenol as well as a small amount of urea. Sweating allows the body to regulate its
temperature. It is controlled from a center in the preoptic and anterior regions of
the Hypothalamus. The volume of water lost in sweat daily is highly variable
ranging from 100 to 8,000ml/ day. The solute loss can be as much as
350mmol/day and sodium under the most extreme condition.

The Composition of Sweat are; water, minerals (i.e. Na + = 0. 9 g/l, K+ = 0. 015g/l.

Mg2+ = 0.0013g/l, Zn2+ = 0.4mg/l, Cu2+ = 0.3 - 0.8mg/l, Fe2+ = 1mg/l, Chromium =
0.1mg/l, Nickel = 0.05mg/l, Lead = 0.05mg/l), lactate and urea.

URINE

This is a typically sterile lipid by-product of the body that is secreted by the
kidneys and voided out through the process called micturition and excreted
through the urethra. Cellular metabolism generates numerous by-products mainly
rich in Nitrogen, that require elimination from the bloodstream. These by-
products are eventually expelled from the body via micturition, the primary
method for excreting water-soluble chemicals from the body. These chemicals
can be detected and analyzed by the procedure called urinalysis. Urine is
produced in the kidney by a process of filtration, reabsorption and tubular
secretion.

Composition of urine: urine is an aqueous solution of >96% of water with the

remaining constituents, in order of decreasing concentration: urea = 9.3g/l,
chloride = 1.87g/l, sodium = 1.17g/l, potassium = 0.750g/l, creatinine = 0.670g/l,
and other dissolved ions, inorganic and organic compounds.

Urine is sterile until it reaches the urethra where the epithelial cells lining the
urethra are colonized by facultative anaerobic Gram-negative rods and cocci.
Some diseases alter the quantity and constituents of urine, such as sugar as a
consequence of Diabetes.

Characteristics of urine: Typically, color can range from clear to a dark amber,
depending mostly upon the level of hydration of the body among other factors.
The following are characteristics of urine.

I. Color: Urine is a transparent solution that can range from colorless to

amber but usually a pale yellow. The color comes primarily from the
 presence of Urobilin. Urobilin in turn is a final waste product resulting from
the breakdown of heme from hemoglobin during the destruction of ageing
Red Blood Cells.

II. Oduor: Smell of urine can be affected by food e.g. eating asparagus is
known to cause a strong Oduor in human urine. This is because of the
body's breakdown of asparagus acid.

III. Turbidity: Turbid (cloudy) urine may be a symptom of bacterial infection,

but can also be caused by crystallization of salts such as calcium phosphate.

IV. PH: The Ph of urine is close to neutral (7) but can normally vary between
4.6 - 8.0. In persons with hyperuricosuria, acidic urine can contribute to
formation of stones of uric acid in the kidney, ureters or bladder.

V. Volume: The amount of urine produced depends upon numerous factors

including level of hydration, activities, environmental factor, weight of
individual and individual health. In adult human, the average production is
about 1 - 2 liters per day. Polyuria is a condition of excessive production of
urine (> 2.5l/day) in contrast to Oliguria where < 400mls are produced per
day or anuria with a production of < 100mls per day.

VI. Specific gravity: Normal value vary between 1.004 - 1.035gcm 3 and any
deviation may be associated with urinary disorders.

Routine Examination of Urine

Specific gravity is normally 1.003 - 1.035, but, in some conditions like chronic
nephritis, it decreases.

Normally, substances like water, salt, amino acids and creatinine are excreted in
urine. But, if abnormally large amount is excreted, it suggests some abnormal
functional status of kidney. If 4 - 5 liters of water is excreted constantly per day, it
is suggestive of diabetes Insipidus.
Abnormal amount of salts or nutritive substances like amino acids appear in urine
during congenital tubular defects. Abnormal Albumin excretion occurs in
defective filtration. Abnormal amount of glucose is excreted in diabetes mellitus.

Microscopic examination: the presence of RBCs, pus cells, epithelial cells, casts
and crystals indicates renal pathology.

HUMAN MILK

This is a fluid secreted by mammary tissues (breast) which is mainly meant for the
nourishing of infants. It is stimulated by the hormone-prolactin. The first secretion
from the breast after delivery is called Colostrum. The Colostrum is a clear fluid
which contains about the same concentrations of proteins and lactose as milk, but
has almost no fat. It contains IgG antibodies. Within two or three days, the
breasts start to secrete large quantities of milk instead of colostrum. In addition
to prolactin, many other hormones i.e. adrenocortical hormones, growth
hormone and parathyroid hormone are required for milk production.

Ejection of milk from the breasts depends on the presence of Oxytocin.

Composition of Human Milk

Human milk contains water (88.5%), fat (3.3%), lactose (6.8%)

Lactalbumin and other proteins (0.4%) and ash (0.2%). The concentration of
lactose in human milk is about 50% greater than in cow's milk but the
concentration of protein in cow milk is about two times that in human milk. Also,
ash, which contains the minerals, is only one-third of that of cow's milk in human
milk.

At the peak of lactation, up to 1.5litres of milk may be formed per day. Since all
the constituents of milk come from the mother's body, maternal nutrition must
be adequate to ensure good quality milk yield and to preserve the health of the
mother.

SALIVA

This is an aqueous solution found in the oral cavity which is produced by the
salivary glands. The major salivary glands consist of paired (22-30%) parotid,
submandibular (60-65%) and sublingual glands (2-4%). Numerous minor salivary
glands (10%) are found in the lower lip, tongue, palate, cheeks and pharynx. The
daily volume of whole saliva secreted by the glands varies between 1 and 1.5L.

Composition of Saliva: The composition of saliva is affected by some factors, such

as diet, genetic effects, age, sex and species. Saliva composition is;

a. Water- 93%

b. Electrolytes; major ones are-Na+, k+, cl-, ca2+, po42-, Hco-3. And those present
in less than 1min/ minor ones are: - F-, Thiocyanate, Mg2+, So-4, I-.

c. Organic compounds i.e. proteins, salivary amylase, blood group substances;

A, B, O, and H, mucus and other glycoproteins, lysozyme, lingual lipase,
epidermal growth factor (EGF) which stimulate mitotic activity and inhibit
 gastric acid secretion. Some trace substances such as urea, uric acid,
glucose, lipids and amino acids.

The most important factor that affects the composition of saliva is the flow rate.
The concentration of Na+, Cl-, and Hco-3 in saliva increase with increasing flow rate
while concentration of potassium is fairly constant over a wide range of flow rate.
During sleep, the flow rate of saliva is nearly zero. This is of high clinical
significance. The highly reduced or absent salivary flow during sleep allows the
rapid proliferation of oral micro-organism and demineralization of the enamel
caused by the acid end products of bacterial metabolism. This is associated with
high incidence of dental caries and other oral cavity diseases. As a result of the
diminished buffering and cleansing activities of saliva during sleep, it is important
to remove before sleep plaque micro-organisms and food debris that form
substrates for the metabolism of the oral bacteria. This diminishes the chances of
developing caries and oral infections.

Dehydration reduces saliva flow rate while over-hydration e.g. by excessive

drinking of water increases saliva flow rate. Gustatory stimulation (taste)
increases salivary flow rate especially stimulation by acidic or flavored substances.
Although olfactory stimuli increase the flow rate of saliva, they are less than 10%
as effective as gustatory stimuli.

The effect of diet on flow rate and composition can be local or systemic. At the
local level, foods that require a lot of chewing or foods that are highly flavored
can increase salivary flow rate and change its composition. At the systemic level,
diets containing a lot of proteins can increase both plasma and salivary urea
concentration. Dietary intake of electrolytes does not affect their concentration in
plasma or saliva because their blood levels are carefully regulated. Moderate
changes in dietary intake of many food items have little or no effect on the
composition of saliva.

Differences have been reported in the protein contents of saliva in Whites, Blacks
and Chinese. Hence, genetical differences influence composition of saliva.

Age and sex affect salivary composition. The flow rate of saliva increases
progressively from childhood to adolescence. In old age, salivary flow rate
decreases. Sexual differences have been found in the flow rate and the
concentrations of Na+ and K+ in saliva. Composition of saliva has also been found
to vary during the female sexual cycle.

Functions of Saliva

1. General cleansing and protection of oral cavity.

2. Starts the digestion of carbohydrate

3. Provides mucous which acts as a lubricant and has some other functions

4. When saliva is absent, it provides the urge to drink

5. It dissolves many components of oral content and contribute to taste

6. By keeping the mouth moist; it helps speech, promotes oral comfort, assist
chewing, aids swallowing

7. Contributes to the digestion of fat.

Protective functions of saliva and constituents of saliva involved.

Protective actions Related constituents

1. PH and buffering capacity Bicarbonate (1%), phosphate, urea, proteins

2. Mechanical cleansing Flow rate, Aggregating factors.

3. Antisolubility. Calcium, phosphate, proteins maintaining

saturated and supersaturated solutions of calcium
phosphate salts, fluoride

4. Antibacterial action. Secretory IgA, Lactoperoxidase-thiocyanate-

hydrogen peroxide system, lysozyme, lactoferrin.

BLOOD

INTRODUCTION: Blood is a connective tissue in fluid form. It carries O 2 from lungs

to all parts of the body and CO2 from all parts of the body to the lungs, hence
referred to as the fluid of life. Also, called the fluid of growth because it carries
nutritive substances from the digestive system and hormones from endocrine
gland to all the tissues. The blood is also called the fluid of health because it
protects the body against diseases and gets rid of the waste products and
unwanted substances by transporting them to the excretory organs like the
kidneys.

PROPERTIES OF BLOOD
1. Color - Blood is red in color. Arterial blood is scarlet red because it contains
more O2 and venous blood is purple red because of more CO2.
2. Volume - The average volume of blood in a normal adult is 5L.In new born
baby the volume is 450ml. It increases during growth and reaches 5L at the
time of puberty. In females it is slightly less and it is about 4.5L.
3. Reaction and PH - Blood is the lightest alkaline and its PH in normal condition
is 7.4
4. Specific gravity - The specific gravity of total blood - 1.052 to 1. 061.
The specific gravity of blood cells - 1.092 to 1.101
The specific gravity of plasma - 1.022 to 1.026
5. Viscosity - Blood is five times more viscous than water due to red blood cells
and plasma proteins.
6. Composition - Blood contains the blood cells called formed elements and the
liquid portion known as plasma.

Three types of cells are present in the blood:

- Red blood Cells or Erythrocytes

- White blood Cells or Leukocytes

- Platelets or Thrombocytes

Plasma - This is blood minus the blood cells. If blood is collected in a hematocrit
tube along with a suitable anticoagulant and centrifuged for 30 minutes at a
speed of 3000 RPM (Revolution per minute), the red blood cells settle down at
the bottom, with clear plasma at the top. The plasma forms 55% and the blood
cells form 45% of the total blood. The volume of red blood cells expressed in
percentage is called the hematocrit value or packed cell volume (PCV).

- Plasma is straw colored clear liquid. It contains 91 - 92 % of water and 8 - 9% of

solids. The solids are the organic and the inorganic substances.

The organic substances include

- Plasma proteins: Albumin, Globulin, fibrinogen

- Amino acids: Essential and non-essential amino acids.

- Carbohydrates: Glucose

-Fats: Triglycerides, cholesterol, phospholipids

- Internal secretions: hormones.

- Enzymes: Amylase, carbonic anhydrase, Acid phosphatase, Alkaline

phosphatase, lipase, Esterase, protease, transaminase.

- Nọn protein nitrogenous substances: Ammonia, Creatine, Creatinine, Xanthine,

Urea, Uric acid, Hypoxanthine.

- Antibodies.

The inorganic substances are: Sodium, Calcium, Potassium, Magnesuim,

Bicarbonate, Chlorides, Phosphate, Iodide, Iron, Copper

FUNCTIONS OF BLOOD
1. Nutritive function: Nutritive substances like glucose, amino acids, lipids and
vitamins derived from digested food are absorbed from the GIT and carried by
blood to different parts of the body for growth and energy production.
2. Respiratory function: Blood carries oxygen from alveoli of lungs to different
tissues and carbon dioxide from tissues to alveoli.
3. Excretory function: Waste products formed in the tissues during various
metabolic activities are removed by blood and carried to the excretory organs
like the kidney, skin, liver etc. for excretion.
4. Defensive function: The white blood cells play important role in the defense of
the body. Neutrophils and monocytes engulf the bacteria by phagocytosis;
lymphocytes are involved in the development of immunity and Eosinophils are
responsible for detoxification, disintegration and removal of foreign proteins.
5. Storage functions: Blood serves as a ready-made source for water and some
important substances like proteins, glucose, sodium and potassium required
by the tissues and are taken from blood during starvation, fluid loss,
electrolyte loss etc.
6. Transport of Hormones and Enzymes: Blood transports hormones to their
target organs/ tissues. It also transports Enzymes.
7. Regulation of Water Balance: Water content of the blood is freely
interchangeable with interstitial fluid and this helps in regulation of water
content of the body.
8. Regulation of Acid-Base balance: The plasma proteins and Hb act as buffers
and help in regulation of acid base balance.
9. Regulation of Body Temperature: Blood maintains the thermoregulatory
mechanism in the body, that is the balance between heat loss and heat gain in
the body because of its high specific heat.

PLASMA PROTEINS

Plasma proteins are the chief solids of plasma. Normal level in blood is 7.0 - 9.0gm
%.

The plasma proteins are: Serum albumin, Serum Globulin and Fibrinogen

SERUM: This is plasma minus fibrinogen. When blood is collected in a container;

coagulation occurs during which fibrinogen is converted to fibrin and the blood
cells are trapped in the fibrin forming the blood clot. After about 45 minutes, a
straw-colored fluid oozes out of the blood clot. This fluid is called Serum.

Serum is different from plasma in its protein content, as it contains only albumin
and Globulin. Serum contains all other constituents of plasma except fibrinogen.

NORMAL VALUES OF PLASMA PROTEINS (in Gm %)

Total proteins = 7.0 - 7.5 Gm%

Albumin: 3.7 - 5.2

Globulins: 1.8 - 3.6

α1 globulin: 0.1 - 0.4

α2 globulin: 0.4 - 0.8

β globulin: 0.5 - 1.2

Y globulin: 0.7 - 1.5

Fibrinogen: 0.2 - 0.4 (200 - 400 mg%)

Albumin/ Globulin Ration

The ratio between plasma level of albumin and globulin is called Albumin/
Globulin (A/G) ratio. It is an important indicator of some diseases involving liver or
kidney. Normal A/G ratio is 2:1

PROPERTIES OF PLASMA PROTEINS

1. Mol. wt.

Albumin: 69,000

Globulin: 156,000

Fibrinogen: 400,000

Thus mol. wt. of Fibrinogen is greater than that of other proteins.

2. Oncotic Pressure: The plasma proteins are responsible for the oncotic or
osmotic pressure in the blood. The osmotic pressure exerted by proteins in the
plasma is called colloidal osmotic pressure, normal is about 25mmHg. Albumin
plays a major role in exerting osmotic pressure.

3. Specific Gravity: Sp. gravity of the plasma protein is 1.026.

4. Buffer Action: The acceptance of hydrogen ions is called buffer action. The
plasma proteins have 1/6 of total buffering action of the blood.
 SEPARATION OF PLASMA PROTEINS

The plasma proteins are separated by the following methods:

a. Precipitation by 'salting out': By this method, different concentrations of salt

solutions are used to precipitate the various fractions of proteins which are
then separated. The salt solutions are:

- Ammonium Sulphate solution and

- Mixture of Sodium Sulphate and sodium Sulphite solution.

Using this method, three proteins have been separated, namely albumin, globulin
and Fibrinogen.

Globulins are precipitated by half-saturation with ammonium Sulphate, whereas

albumins are precipitated only on full saturation. Fibrinogen is best precipitated
by 1/5th saturation with ammonium Sulphate. Among the Globulins, there is a
fraction which can be precipitated by 1/3 and is termed Euglobins and the rest is
termed Pseudoglobins (false globulins).

Note: Euglobulins (true globulins) are precipitated by 1/3rd saturation of

ammonium Sulphate and are insoluble in distilled water but soluble in dilute salt
solutions (e.g. NaCl). Pseudo globulin (false globulins) is soluble in distilled water.

b. Cohn's Fractionation (using Ethanol)

By this method, Cohn used varying concentrations of Ethanol at low

temperature to separate out fractions of proteins which are called fraction I, II
etc. Each fraction is itself a mixture of proteins but contained one of the
proteins predominantly.
 Fraction I is rich in Fibrinogen

Fraction II is Y globulins

Fraction III contains α and β globulins including Isoagglutinin and Prothrombin

Fraction IV contains α and β Globulins

Fraction V contains predominantly Albumin

Advantage

- Solvent use in the procedure can be readily removed by evaporation.

- Mild procedure adopted in fractionation of the proteins do not cause
denaturation of the proteins.

Clinical use: Method is useful for obtaining purified proteins on a large scale
for therapeutic purposes.

c. Electrophoresis: More recent methods include separation of plasma

proteins by paper electrophoresis (Tiselius, 1937), gel electrophoresis.
In this method, plasma proteins are separated depending on their differences
in electric charge and the rate of migration. It is done in a Tselius apparatus by
using paper or cellulose or starch block. By this method, the proteins are
separated into albumin (55%), alpha globulin (13%) β globulin (14%), Y
(gamma) globulin (11%) and Fibrinogen (7%)
d. Ultracentrifugation: Separates albumin, globulin and Fibrinogen depending
upon their density. The method is also useful in determining the molecular
weight of these proteins.
e. Immunoelectrophoretic method: Using this method, the proteins are
separated on the basis of electrophoresis patterns formed at the site of
antigen - antibody reactions.

CHARACTERISTICS OF INDIVIDUAL PLASMA PROTEINS

A. ALBUMIN: It is the most abundant and fairly homogenous protein of plasma.

Has a mol. of 69,000. Approximately half of the total protein of plasma is
albumin. It has low iso-electric PH (PI = 4.7). The protein migrates fastest in
electrophoresis at alkaline PH and precipitates last in ‘salting out’ or alcohol
precipitation methods. It is a simple protein, consists of a single polypeptide
chain, having 585 amino acids, with 17 inter-chain disulfide (s - s) bonds. The
protein is precipitated with full saturation of ammonium Sulphate (NH 4)2SO4.

Site of synthesis: Albumin is mainly synthesized in liver. Rate of synthesis is

approximately 14.0 Gms/day

Clinical importance: Decrease in Albumin concentration is seen in severe

protein energy or calorie malnutrition (PEM or PCM), liver diseases like
cirrhosis of liver (albumin synthesis is impaired), nephrotic syndrome (Albumin
is lost in urine). Decrease in albumin concentration leads to oedema
formation. Oedema occurs when total proteins fall below 5.0gm% and albumin
level below approximately 2.5gm%.

FUNCTIONS OF ALBUMIN
 - It exerts low viscosity

- Contributes 70 to 80% of Osmotic pressure and plays an important role in

exchange of water between tissue fluid and blood.

- Also undergoes constant exchange with Albumin present in extracellular

spaces of muscles, skin and intestines.

- Helps in transport of several substances viz. FFA, unconjugated bilirubin,

Ca++and steroid hormones.

- Certain drugs also bind to Albumin, e.g. Sulphonamides, aspirin, penicillin,

etc. and are transported to target tissues.

- Nutritive function - Albumin in plasma is in a dynamic state with a rapid

turnover with a specific half-life. It is delivered to cells where it is
hydrolyzed and cellular proteins are synthesized.

B. GLOBULINS: They are separated by half saturation with ammonium

Sulphate. Mol. weight ranges from 90,000 to 1,300,000. By electrophoresis,
globulins can be separated into different fractions viz α1 globulins, α2
globulins ,β globulins and Y-globulins.

Site of synthesis: α and β globulins are synthesized in the liver; Y-globulins

are synthesized by plasma cells and β-cells of lymphoid tissues (RE system).

1. α- Globulins - they are glycoproteins and are further classified into α 1

and α2 depending on their electrophoretic mobility.
 a. α1 - acid glycoprotein - Also called "Oroso-mucoid". Concentration in
normal plasma is 0.6 - 1.4gm per liter (average = 0.9). It's carbohydrate
content is about 41%.

- Considered to be a reliable indicator in acute inflammation.

Clinical importance

1. Increase - consistently rises in acute and chronic inflammatory diseases, in

cirrhosis of the liver and in many malignant conditions.

2. Decrease - low concentrations (abnormally low) occurs in generalized hypo

proteinaemic conditions viz hepatic diseases, Cachexia, malnutrition and
nephrotic syndrome.

b. α1-fetoglobulin (α1-fetoprotein) - present in high concentration in fetal blood

during mid pregnancy. Normal adult blood has less than 1 micro gram/100ml.
Presence of α1-fetoprotein is useful diagnostically in determining presence of
hepatocellular carcinoma or teratoblastoma ("tumor marker")

II. α2-Globulins - comprises Caeruloplasmin and Haptoglobulin. Normal plasma

contains about approximately 30mg/100ml and about 75-100ug of Cu may be
present in 100ml of plasma. Caeruloplasmin is a copper containing α 2- globulin, a
glycoprotein with enzyme activities e.g copper oxidase, histaminase and ferrous
oxidase. Mol. wt is 151,000. It has eight sites for binding copper.

Site of synthesis: it is synthesized in the liver when 8 copper atoms are attached
to a protein, "apocaeruloplasmin".
Level of Caeruloplasmin with Age and Sex: There is low concentration at birth,
gradually increases to adult levels and slowly continues to rise with age
thereafter. Adult females have higher concentration than males.

Clinical importance: Increase is found in pregnancy, inflammatory processes,

malignancies, oral estrogen therapy and contraceptive pills.

Decrease in Wilson's ds and in Menke's dz.

Function: It mainly functions as a ferroxidase and helps in oxidation (conversion)

of Fe++ to Fe+++ which can be incorporated in transferrin. Haptoglobulin is
synthesized principally in liver by hepatocytes and to a very small extent in cells of
R. E cells. It is comprised of two kinds of polypeptide chains, two α-chains
(possibly three) and only one form of β-chain.

C. α1-globulin-inhibitors: some of the α1-globulins act as inhibitors of coagulation

and also inhibit some digestive enzymes like trypsin and chymotrypsin.

D. α1-antitrypsin (α-AT) - It is α1-antiproteanase, molecular weight approximately

45,000 to 54,000. Isoelectric point Pl is 4.0. Normal value in adults is 2 to
4gm/liter (approximately 290mg/ml). It is synthesized by liver and it is the
principal protease inhibitor (Pi) of human plasma. It inhibits trypsin, elastase and
certain other proteases by forming complexes with them.

Increase of serum α1-antitrypsin is seen in response to inflammation, acute,

subacute and chronic. It is considered as one of the acute phase reactants and
increased in trauma, burns, infarction, malignancy and liver disease etc. Chronic
hepato-cellular diseases and biliary obstruction, either normal or increased. In
pregnancy and also during contraceptive medication. Neonates have serum
concentrations much below adult values, but show gradual increase with age and
achievement of adult levels is rapid.

Decrease in Serum α1-antitrypsin: In protein losing disorders, e.g. nephrotic

syndrome, diffuse hypoproteinemia, in emphysema of lungs and in juvenile
cirrhosis of liver.

Clinical significance

1. Role in Emphysema lung (An abnormal accumulation of air in tissues especially

the lungs). Pulmonary emphysema is a chronic lung disease, characterized by an
abnormal increase in size of the air spaces, resulting in labored breathing and
caused by exposure to toxic chemicals such as tobacco smoke. A deficiency of α 1-
AT has a role in certain cases approximately 5% of emphysema of lung. This
occurs mainly with ZZ phenotype who synthesize Piz

Biochemical mechanism:

Normally, α1-AT protects the lung tissues from injurious effects by binding with
the proteases, viz active protease. A particular methionine (358 residue) is
involved in binding with the protease.

Thus, Active elastase + α1-AT

Inactive elastase: α1-AT complex

No proteolysis of lung No tissue damage

When α1-AT is deficient or absent, the above complex with active elastase does
not take place and active elastase brings about proteolysis of lung and tissue
damage.

Active elastase + No or decreased α1-AT

Active elastase proteolysis of lung

(complex does not form) Tissue damage

2. Relation of Smoking with Emphysema: Smoking oxidizes the methionine (358

residue) of α1-AT and thus inactivates the protein, hence such α 1-AT molecule
cannot bind to the protease active elastase and thus proteolysis of lung and tissue
damage occurs, accelerating the development of emphysema.

TREATMENT- IV Adm of Α1-AT in patients with emphysema due to α 1-AT

deficiency.

Recently, by protein engineering to replace methionine at 358 by another residue

that would not get oxidized by smoking. The resulting mutant new α 1-AT can
afford protection against the protease by inactivating it. Attempts are being made
to develop gene therapy for emphysema due to Α1-AT deficiency.

3. Role in Cirrhosis: Juvenile hepatic cirrhosis has also been correlated with α 1-AT
deficiency. In this condition molecules of Pi z (ZZ phenotype) accumulate and
aggregate in the cells of the cisternae of the endoplasmic reticulum of
hepatocytes. The hepatocytes cannot secrete this particular type of α 1-AT. Thus,
Piz protein of α1-AT is synthesized but not released from the hepatocytes. Thus,
there is aggregation due to formation of polymers of mutant α 1-antitrypsin (the
polymers forming as a result of strong interaction between a specific loop in one
molecule and a prominent B-pleated sheet in another, called Loop-sheet
polymerization). The aggregates lead to damage to liver cells leading to hepatitis
and Cirrhosis.

4. Role as a tumor maker: α1-AT has been used as a tumor maker. It is increased
in germ cell tumors of testes and ovary.

5. As an inhibitor of fibrinolysis: α1-AT is one of the most important inhibitors to

fibrinolysis along with α1-antiplasmin and α2-macroglobulin. All these inhibitors
block the action of plasmin on fibrinogen.

Functions of Haptoglobulin

- The function of haptoglobulin is to bind free Hb by its α-chain and minimizes

urinary loss of Hb. Abnormal Hb such as Barts- Hb and Hb-A have no α-chains and
hence cannot be bound.

-After binding, Hp - Hb complex circulates in the blood, which cannot pass

through glomerular filter and ultimately the complex is destroyed by RE cells.

III. β -Globulins - Consists of β -lipoproteins (LDL) and Transferrin.

Transferrin is a non-heme iron containing protein formerly called siderophilin.

Exists in plasma as B1-globulin, a glycoprotein with Mol. wt. 70,000 and it is a true
carrier of Fe. Protein Part is Apo-transferrin and can bind two atoms of Fe to form
"Transferrin".
Site of synthesis: the protein synthesized principally in the liver. There is also
evidence of its synthesis in bone marrow, spleen and lymph modes, perhaps by
lymphocytes.

Clinical importance: Increase serum transferrin levels, are seen in iron def.
anemia and in last months of pregnancy.

Decrease in Cirrhosis of liver, nephrotic syndrome, acute illness such as trauma,

myo-cardial infarction and malignancies.

Functions

1. Transport of Fe between intestine and site of synthesis of Hb and other Fe

containing proteins.

2. It has been seen that unsaturated transferrin has a bacteriostatic function

which is attributed to sequestration of Fe required by micro-organisms.

C-reactive proteins: It is present in concentration less than 1mg/100ml in the

adult male

It precipitates with group c polysaccharides of pneumococci, in the presence of

Ca++, hence, called "c-reactive protein".

Two electrophoretic forms appear on immunoelectrophoresis:

. CPR alone in the Y-band

. A CPR complex with an acidic mucopolysaccharide termed m-cPR in the β -

region.

Functions
. It can bind heme.

. It can bind to T-lymphocytes and can activate complement.

Hemopexin: It is a β -globulin and migrates electrophoretically in the globulin

region in the electrophoretogram. Mol. wt. 57,000 to 80,000. Normal value in
adults = 0.5 to 1.5gm/l. The level is very low at birth but reaches adult value
within the first year of life. It is synthesized by parenchymal cells of liver.

Function

The principal function is to bind and remove circulating haem which is formed in
the body from breakdown of Hb, Myoglobin or catalase. It binds heme and
several other porphyrins in 1:1 ratio. The haem-hemopexin complex is removed
by the parenchymal cells of liver.

Clinical significance

Decrease is seen in: I. Hemolytic disorders; ii. At birth in new born; iii.
Administration of diphenylhydantoin.

Increase is seen in: i. Pregnancy; ii. In diabetes mellitus; iii. Duchenne muscular
dystrophy; Iv. Some malignancies especially melanomas.

Complement C1q

Complement is a collective term for several plasma proteins that are precursors
to certain active proteins circulating in blood. These proteins participate in
immune reactions in the body. After the formation of immune complex, C1q is the
first complement factor that is bound. The binding takes place at the 'Fc' or
constant part of the IgG or IgM molecule. The binding triggers the classical
complement pathway.

It is thermo labile and is destroyed by heating at 56 oc for 30 mins; normal value in

adult is 0.15gm/l; mol. wt 30,000. It can bind heparin and bivalent ions such as Ca +
+
.

Clinical significance

Decrease in plasma C1q is used as an indicator of circulating antigen-antibody

complexes. Thus, levels are decreased in active immune diseases such as SLE; in
disorder of protein synthesis and in increased protein loss in intestinal diseases.

Increase: levels are increased in certain chronic infections and in rheumatoid

arthritis.

IV. B2 - MICROGLOBULINS

It is a low mol. wt. peptide containing 100 a-a residues and is excreted in urine. It
is present in urine to the extent of only 0.01mg/100ml. It also has close structural
resemblance to immunoglobulins.

Urinary β 2-M level is elevated in Wilson's ds (Hepatolenticular degeneration),

renal diseases like chronic nephritis, chronic cadmium poisoning, Fanconi
syndrome.

Serum B2-M is elevated in chronic nephritis, myelomatosis, systemic lupus

erythromatosis (SLE), malignant tumors, abnormal pregnancies, chronic
rheumatism and Behcet syndrome.

ACUTE PHASE Proteins (or Reactants)

Levels of certain proteins in plasma increase during acute inflammatory states or
secondary to certain types of tissue damage. These proteins are called Acute
phase proteins or Reactants. They include -

. C-reactive protein (CRP); Haptoglobulin (Hp)

. α1-antitrypsin; α1-acid glycoprotein (Oroso-mucoid) and Fibrinogen

. V - Y-Globulins

These are immunoglobulins having anybody activity.

C. Fibrinogen: Is a soluble glycoprotein, also called clotting factor 1, as it takes

part in the coagulation of blood and it is the precursor of fibrin, the substance
required for clotting. Normally constitutes 4-6% of total protein of blood. It is
precipitated with 1/5th saturation with ammonium Sulphate. Molecular wt ranges
between 350,000 - 450,000; being asymmetrical and large, it is important for
viscosity of blood.

Site of synthesis: All the three chains are synthesized in the liver. Three structural
genes involved in the synthesis are on the same chromosome and their
expression are regulated coordinately in humans.

Structure: It is made up of 6 polypeptide chains, 2Aα,2B β and 2 Y-chains, thus

forming Aα2BB2Y2. Chains are linked together lengthwise by S-S linkages.

Other proteins of clinical interest

1. Bence-Jones Protein
It is defined as monoclonal light chains present in the urine of patients with
paraproteinemic states. Either monoclonal 'k' or 'λ' light chains are excreted in
significant amounts in about 50% cases of multiple myeloma. That is, it is an
abnormal protein found in the blood and urine of people suffering from a disease
called multiple myeloma (a plasma cell tumor). It has mol. wt 45,000.

Identification - Heat test.

The protein is identified easily in urine by a simple Heat test. On heating the urine
to 50oC to 60oC, Bence- jones proteins are precipitated; but when heated further,
it dissolves again. Reverse occurs on cooling. Best detected by zone
electrophoresis and immunoelectrophoresis of concentrated urine.

2. Cryoglobulins

These are proteins which are coagulated when plasma or serum is cooled to very
low temperature (2 to 4 0c). Most commonly they are monoclonal 1gG or 1gM or a
mixture of two. These are present even in normal individuals. Mol. wt. vary from
1,650,000 to 6,000,000.

Increase - In rheumatoid arthritis, lymphocytic leukemia, multiple myeloma,

lymphosarcoma’s and SLE.

FUNCTIONS OF PLASMA PROTEINS

1. Nutritive: They are simple proteins and a good source of proteins. They are
useful in hypoproteinaemic states. They contribute amino acids for tissue protein
synthesis.
2. Fluid Exchange: They play an important role in the distribution of water
between the blood and tissues. This is because of the colloid osmotic pressure of
plasma proteins.

At the arterial end of capillary; hydrostatic pressure (tends to drive out fluid)
exerted is greater than the osmotic pressure. Net filtration pressure is 7mmHg
which drives the fluid out from the vessels to tissue spaces. On the other hand, in
the venous end of capillary loop, osmotic pressure (tends to draw in fluid) is
greater than the hydrostatic pressure and net absorption pressure is 8mmHg
which draws fluid from tissue spaces into the vessel.

This explanation of the mechanism of exchange of fluids and dissolved materials

between the blood and tissue spaces is called the "starling hypothesis".

3. Buffering action: Like other proteins, the serum proteins are amphoteric, and
thus can combine with acids or bases. In acidic PH, NH 2 group acts as base and can
accept a proton and thus is converted to NH3+ and in alkaline PH, COOH group acts
as acid and can donate a 'proton' and thus have COO-

4. Role in Transport Mechanism: Plasma proteins are essential in the transport of

various substances in the blood. Albumin, α and β globulins are responsible for
the transport of the hormones, enzymes etc. The α and β globulins play an
important role in the transport of metals in the blood.

5. Roles as Reserve Proteins: During fasting, inadequate food intake and

inadequate protein intake, the amino acids from plasma proteins can be taken up
by tissues and used for building up new tissue protein. Thus, plasma proteins
during these conditions are utilized by body tissues as the last source of energy,
hence are called the reserve proteins.

6. Role in Viscosity of Blood: Due to the presence of proteins, blood is a viscous

liquid. Globulins and Fibrinogen which are large in size and asymmetrical account
for the viscosity of blood. The viscosity of blood provides resistance to flow of
blood in the blood vessels to maintain blood pressure in the normal range.

7. Immunological function (Body defense): The gamma globulins play an

important role in the defense mechanism of the body by acting as antibodies
(immune substances) and protect the body against microbial infections.

8. Role in Blood Coagulation and Fibrinolysis: In addition to prothrombin and

Fibrinogen, plasma contains a number of other components, enzymes and clotting
factors, which participate in the process of coagulation of blood. Thrombus, an
intravascular clot whenever it is formed is digested by the enzymes of fibrinolytic
system present in plasma, then preventing thrombosis.

HEMOGLOBIN (Hb)

Introduction:

Hemoglobin (Hb) and Myoglobin are heme proteins which are essential for O 2
supply for metabolism.

Hemoglobins are globular proteins, present in high concentrations in red blood

cells that bind oxygen in lungs and transport it to the tissues or cells. They also
transport or retrieve CO2 and Protons (H+) from the tissues to the lungs, and they
carry and release nitric oxide (NO) in the blood vessels of the tissues. NO is a
potent vasodilator and inhibitor of platelet aggregations. Whereas hemoglobin is
tetrameric, Myoglobin is Monomeric and functions basically to store oxygen as a
reserve against oxygen (O2) deprivation. That is, in contrast to hemoglobin which
has four chains and four O2-binding sites, myoglobin contains only a single
polypeptide chain and one O2-binding site.

Heme - Containing proteins are characteristic of the aerobic organisms, and are
altogether absent in anaerobic forms of life. Normal concentration of Hb in an
adult male varies from 14.0 to 16.o gms % and there are about 750gms of Hb in
the total circulating blood of a 70kg man.

The structure of Heme remains the same in Hb from any animal source, the basic
protein Globin varies from species to species in its amino acid composition and
sequence and thus responsible for the species - specificity.

The polypeptide chains of Globin of adult Hb, are characterized by a relatively

high content of "histidine" (His) and "lysine" (Lys) and a small amount of
"Isoleucine" (Ile).

STRUCTURE OF HB

The structure of Hb molecule can be discussed under two headings:

i. Structure of Heme, the Prosthetic group, a non-polypeptide moiety that forms

functional part of a protein, and

ii. Structure of Globin, the protein part - apoprotein.

(Note - with its prosthetic group, it is a Holoprotein)

1. HEME
It is a Fe-Porphyrin compound with a tetra-pyrrole structure or ring. Each Pyrrole
ring has the following structure.

HC CH

HC CH

NH Pyrrole ring.

Four such Pyrroles called I to IV are linked through - CH= bridges called Methyne
or Methylidene bridges to form a porphyrin nucleus.

M – C1 C2 – V M – C3 C4 – V
α
C I C H C II C

N C N M = Methyl – CH3
 V = Vinyl – CH = CH2

ᵟ CH Fe2+ H 2O CHβ P = Propionic acid ----

CH2 – CH2 ~ COOH

N N
ϒ
C IV C CH C III C

M – C8 C7 – P P – C6 C5 – M

Lys His Arg

Globin

Structure of Heme

- The outer carbons of the four pyrrole rings which are not linked with the
methylidene bridges, are numbered 1 to 8

- The Methylidene bridges are referred to as α, β, ϒ and ϭ respectively.

- The two hydrogen atoms in the - NH groups of the pyrrole rings (II and IV) are
replaced by Ferrous ion (Fe++) which occupy the center of the compound ring
structure and establish linkages with all the four nitrogens of all the pyrrole rings.

- The Fe besides its linkages to four nitrogens of the pyrrole rings, is also linked
internally (5th linkage) to the nitrogen of the imidazole ring of histidine, called the
Proximal histidine (F8) or 87 (in hemoglobin and Myoglobin structures) of the
polypeptide chains ("heme-linked" group). It is considered to have a valency of six
as in Ferrocyanide H4Fe (CN)6. O2 forms the sixth bond; the O 2 is located between
the ferrous atom and a second histidine imidazole, designated the distal histidine
(E7 or 58). In deoxyhemoglobin, the sixth position is unoccupied or linked to a
molecule of water. When Hb is oxygenated, the H2O is displaced by O2

Hb. H20 + O2 Hb. O2 + H2O

- The propionic acid COOH groups of 6 and 7 positions of heme of III and IV
pyrroles are also linked to the amino acids Arg and Lys of the Polypeptide chains.

- The hydrogens at positions 1 to 8 are substituted by different groups in different

compounds. In Protoporphyrin IX, which forms parent compound of heme, the
positions 1 to 8 in Pyrroles are substituted by methyl (-CH 3), Vinyl (-CH = CH3),
Methyl, Vinyl, Methyl, Propionic acid (-CH 2 - CH2 - COOH), Propionic acid and
Methyl groups in that order respectively.

2. GLOBIN: Globin, the protein part of Hb, is composed of four polypeptide

chains, two identical α - chains and two identical β - chains in normal adult Hb.

Each polypeptide chain contains a 'heme', in the so-called "heme-pocket". Thus,

one Hb molecule contains four heme units.

The Alpha (α) chain contains 141 amino acids, whereas the Beta ( β), Gamma (r)
and Delta (D) each comprises of 146 amino acids. The α and β subunits are held
together by hydrogen bonds and Vander Waal Forces.

Note: Electrostatic bonds which occur within subunits and also between subunits
help in ensuring conformational stability.

(The bonds are Inter and Intra)

There are four (4) heme molecules per Hb-molecule i.e. each sub unit has 1 . The
heme is found within the clefts in Helices that occur in the Globin chain.
Hemoglobin (Hb) has seven (7) helices in its polypeptide chain, numbered A-G and
the heme is located in the cleft between Helices E and F. Each Hb molecule
contains 38 Histidine (His) residues which play a role in the buffering action of Hb.

Heme

A B C D E F G

The Iron atom (Fe) component of Haeme is usually centrally located in the
porphyrin ring and this Iron which is the Ferrous State (Fe 2+) possesses a total of
six (6) valences.

In oxygenated Hemoglobin (Hb), the 6th valency is what binds the oxygen (O 2).
The other (5) valences are distributed as follows:

- Four (4) to the pyrrole Nitrogen

- The 5th to the imidazole Nitrogen of the Proximal Histidine.

Note: Ferric Iron (Fe3+) has five (5) valences.

A Hemoglobin (Hb) binds 4 molecules of Oxygen, O 2. The association of chains

into a tetrameric structure as seen in Hemoglobin results in a better efficiency in
Oxygen transfer than would occur in single chains.

Varieties of Normal HB: Normal human Hb comprises of three forms, namely: -

a. Hemoglobin A1 (HbA) (major Adult Hb) - consists of a pair of Alpha (α) and a
pair of Beta (β) chains i.e. α2 β2
b. Hemoglobin A2 (HbA2) (Minor Adult Hb) - consisting of a pair of alpha (α) and a
pair of Delta (D) chains i.e. α2δ2

C. Hemoglobin F (Fetal - Hb) - consisting of a pair of alpha (α) and a pair of

Gamma (ϒ) chains i.e. α2 ϒ2

- Hb A accounts for about 97% of total adult Hemoglobin in a healthy subject.

- Hb A2 and F accounts for approximately 2% and less or equal to (≤1%) one

percent respectively.

(Alpha (α) chain gene is on chromosome 16 while the Beta (β) gamma (ϒ) and
Delta (D) chains are on chromosome 11-)

d. Embryonic Hb: This is found in first three months of intrauterine life of the baby
(pregnancy). It contains two α-chains and two Ɛ-chains; thus, it is α 2Ɛ2. They
include Gower I and Gower II.

e. Hb - A3 - Is an altered form of Hb-A. Found chiefly in old red cells. (3 - 10% of

total)

f. Hb - A1c (Glycosylated Hb i.e. glucose molecules in blood that get stuck to

hemoglobin molecule):- In addition to Hb-A 1, the major form of normal adult Hb,
a minor Glycosylated form is also found in adult red blood cells. In normal
individuals, it is present in concentration of 3 - 5% of total Hb. However, in
patients with diabetes mellitus, it may be increased to as much as 6 - 15% of total
Hb.

HAEMOGLOBIN IN O2 AND CO2 TRANSPORT

1. Combination or Transport of O2:

The physiological importance lies in the fact that Hb can readily combine with O 2.
Hb binds 4 molecules of O2 per tetramer and the binding of one molecule leads to
increase affinity or binding of further O 2 molecules. This phenomenon is called co-
operative binding.

The combination is loose and reversible. The gas is taken up readily at high partial
pressures e.g. in lungs and released readily at low O 2 pressures e.g in tissues; thus
providing an effective system for transport from the atmosphere to the cells of
the body.

De-Oxygenated Hb can loosely combine with O 2 forming Oxy – Hb, the

attachment with oxygen occurs with Fe in the heme portion. Iron (Fe) remains in
the ferrous state both in de-oxygenated Hb and Oxy-Hb. (O 2 remains attached
with the unpaired electrons of Fe).

Each heme can bind only one molecule of O 2. Since each molecule of Hb contains
4 mols of heme; hence one molecule of Hb can maximally combine with four (4)
mols of O2.

Factors: The combination is loose and reversible and governed by the following
factors.

- Partial pressure of O2 (Po2) favors Oxygenation

- Partial Pressure of CO2 (Pco2) favors dissociation

- Ph of the medium-acidosis favors liberation of O2

Combination of O2 with one heme situated on α-chain, which can permit a

molecule of O2 to enter, brings about conformational changes, so that O 2 can
enter into other heme groups situated on β chains, (the valine residue is removed
permitting O2 to enter).

The increase in O2 affinity by heme group on β sub-unit after combination of O 2

with α-subunit is called "Heme-Heme interactions"

Hb can occur in two states:

R state - This is the relaxed state and it is oxygenated Hb

T state - This is the tense/ taut state and it is de-oxygenated Hb

2. Combination or transport of Co2

In addition to its role in O2 transport, Hb also transports CO2 and protons (H+)
from the peripheral tissues to the lungs. Hb transport of CO 2 is in form of
Carbamates giving rise to Carbamino-Hb.

CO2 occupies a different binding site on the Hb, binding to the α - NH 2 (amino)
group while protons bind at various places on the protein. CO 2 is more readily
dissolved in de-oxygenated blood facilitating its removal from the body after O 2
has been removed to tissues undergoing metabolism. The increased affinity of
CO2 by the venous blood is known as HALDANE EFFECT. Because of the action of
the enzyme carbonic anhydrase, CO 2 reacts with H2O to form carbonic acid, H 2CO3
which decomposes to HCO3 and protons (H+) . As such, blood high in CO 2 level is
lower in PH.

Deoxy-Hb binds one proton (H+) for every two O2 molecules released. It is of note
that the binding of CO2 and protons to Hb causes a conformational change in the
protein and facilitates the release of O 2. The decrease in Hb affinity for O2 by the
binding of CO2 and proton (H+) is known as BOHR EFFECT.

Conversely, when the CO2 level in the blood decrease, e.g. in the lung capillaries,
CO2 and protons (H+) are released from Hb increasing the O 2 affinity of the
protein.

O2 - Hb Dissociation or O2 Saturation Curve of Hb

The O2 - Hb dissociation curve is sigmoidal and this is as a result of the co-

operative binding of O2 to Hb
 100

90

80

70
Percentage saturation

60

50

40

30

20

10

10 20 30 40 50 60 70 80 90 100
Po2 (mmHg)
O2 - Hb Dissociation curve.

Certain factors ensure or facilitate the effective transport of O 2 from the lungs to
the tissues and carriage of CO2 from the tissues to the lungs. These factors include
PH, 2, 3BPG and temperature. The effect of these factors lead to shifts of the
Normal curve.

A fall in PH (acidosis), rise in temperature and increase in 2,3 BPG lead to the shift
of the curve to the right, meaning or resulting in increased O 2 delivery to the
tissues. The converse occurs when the PH rises (alkalosis), fall in tempt, decrease
conc. of 2,3 BPG, leading to reduced O2 delivery to tissue.

The effect of these allosteric regulators of O 2 affinity of Hb is most evident when

the O2-Hb dissociation curve of Hb is stripped of all its allosteric effects is
compared with the curve pattern when they are there.
 Stripped hb Tempt.

PH
% Saturation

2, 3 BPG

CO2

Po2

Thus, the presence of both Co 2 in the tissues and BPG in the RBCs, creates a
situation in which O2 is effectively transported from lungs to tissue. The Hb
tetramer binds one molecule of 2,3BPG in a pocket in the center of Hb molecule
which is only present in the T-form of Hb. BPG forms salt bridges at the terminal
amino-groups of the two β - chains and thus stabilizes Deoxy-Hb and effectively
reduces the O2 affinity. For the structural transition from T to R state to take
place, the bonds between Hb and 2,3BPG must be broken and the 2,3 BPG
expelled.

ABNORMAL HAEMOGLOBIN

Some forms of Variant Hb molecules exist and most result from genetic mutations
involving the genes that encode the α and β subunits of Hb. About 1000
mutations causing Mutant Hbs are known but many of these Variant Hbs do not
cause abnormalities clinically.
When a mutation leads to the production of an abnormal Hb which affects the
biological function of Hb, it is called Haemoglobinopathy. Some of these Variant
Hbs include Hbs, HbM, HbC, HbD, HbE, Hb Kansas, Hb Chesapeake.

- Hbs: Is due to the replacement of the polar Glutamic acid (Glu) by Valine (Val) at
position 6 of the β chain sub-unit.

- HbC: Is due to Glutamic acid (Glu) replacement by Lysine (Lys) at position 6 of

the β - subunit

HbD: Is due to replacement of glutamic acid (Glu) by glutamine (Gln) on position

121 of the β-chain.

- HbE: Is due to replacement of Glutamic acid (Glu) by Lysine (Lys) on position 26

on the β - chain.

- HbM: Is due to substitutions by Tyrosine which can involve the α or β - chains at

the proximal or Distal Histidine residues. e.g.:

HbM - Boston - Tyrosine replaces Histidine at position 58 on the α-chain

HbM - Hyde Park - Tyrosine replaces Histidine at position 92 on the β-chain.

As stated, some of these Hb Variants do not produce clinical conditions but some
do. Those associated with clinical conditions include:

1. Hbs and HbC - These produce sickle syndrome in the homozygous state with
consequent hemolytic anemia. Hbcc produce mild to moderate hemolytic anemia
while HbAc and HbAs (sickle trait) do not have clinical manifestations.
2. HbM and Hb Kansas - This Hb has low affinity for O 2 and thus produces cyanosis
and the degree varies depending on whether the defect is on the alpha (α) or
Beta (β) Histidine (His) residue.

No hemolytic anemia is associated with HbM and Hb Kansas unlike Hbs and HbC.

3. Hb Chesapeake: This variant Hb has increased affinity for O 2 and thus has
decreased ability to deliver or release O 2 to the tissues. This leads to Hypoxia and
consequent Polycythemia.

4. HbE: Heterozygous are asymptomatic but Homozygous have slight decrease in

Hb - level due to slight increase in RBC fragility.

Apart from mutations leading to substitution of the usual amino acid by another
in the Hb-chains, there could be impairment of synthesis of a single kind of Hb-
chain, that is alpha (α) or Beta (β) chains.

In Thalassemia, the gene function is abnormal but there is no abnormality in the

polypeptide chain sequence. Reduction in alpha (α) chain synthesis is called Alpha
(α) _ Thalassemia while deficient β-chain synthesis is called Beta (β) Thalassemia.

BLOOD COAGULATION

Definition: Coagulation or clotting is the process in which blood loses its fluidity
and becomes a jelly like mass few minutes after it is collected in a container or
shed out. The clot is a mesh of thin fibrils, which consists of fibrin formed from
fibrinogen.

FACTORS INVOLVED IN BLOOD CLOTTING

Coagulation of blood or clotting occurs through a series of reactions due to the
activation of a group of substances, called clotting factors.

Thirteen clotting factors are identified

Factor I - Fibrinogen

Factor II - Prothrombin

Factor III - Thromboplastin (tissue factor)

Factor IV - Calcium

Factor V - Labile factor (proaccelerin or Accelerator globulin).

Factor VI - Presence has not been proved

Factor VII - Stable factor

Factor VIII - Antihemophilic factor (Anti hemophilic globulin)

Factor IX - Christmas factor

Factor X - Stuart-prower factor

Factor XI - Plasma Thromboplastin antecedent

Factor XII - Hegman factor (contact factor)

Factor XIII - Fibrin stabilizing factor (fibrinase)

Each of these factors was named after the scientist who discovered them or as
per the activity. However, Christmas factor was named after the patient in whom
it was discovered.

BLOTTING CLOTTING: The formation of blood clots is the result of a series of

zymogen or proenzyme activations. Normally all the factors are in inactive
proenzyme forms. The amplification achieved by this cascade of enzymatic
activations allow blood clotting to occur rapidly in response to injury. It is carried
out by series of proenzyme-enzyme conversions. The first one of the series is
converted into an active enzyme that activates the second one, which activates
the third one; this continues till the final enzyme thrombin is formed.

In general, clotting of blood occurs in three steps:

1. Formation of Prothrombin factor

2. Conversion of Prothrombin into thrombin

3. Conversion of fibrinogen into fibrin.

Note: Seven of the clotting factors in their active form are serine proteases:
Kallikrein, XIIa, XIa, IXa, VIIa, Xa, and thrombin.

Stage I: Formation of Prothrombin Activator

Prothrombin activator is formed in two ways:

A. The Intrinsic Pathway - Instigated when the blood comes into physical
contact with abnormal surfaces caused by injury i.e initiated by platelets
(which are within the blood itself) which releases phospholipid in contact
with collagen.

B. The Extrinsic pathway is initiated by factors released from injured tissues i.e
tissue Thromboplastin.

INTRISIC PATHWAY FOR THE FORMATION OF PROTHROMBIN ACTIVATOR

I. Following injury, the blood vessel is ruptured. The endothelium is damaged

and collagen beneath the endothelium is exposed

II. When factor XII (Hegman factor) comes in contact with collagen, it is
converted into activated factor XII (XIIa) in the presence of Kallikrein and
high molecular weight (HMW) Kinogen.

III. The activated factor XII converts factor XI into activated factor XI in the
presence of HMW Kinogen.
IV. The activated factor XI activates factor IX in the presence of factor IV
(Calcium).

V. Activated factor IX activates factor X in the presence of factor VIII and

calcium.

VI. Activated factor X reacts with platelet phospholipid and factor V to form
Prothrombin activator in the presence of Calcium ions.

Note: Factor V is also activated by positive feedback effect of thrombin.

EXTRINSIC PATHWAY FOR THE FORMATION OF PROTHROMBIN ACTIVATOR

i. After injury, the damaged tissues release tissue Thromboplastin. The

Thromboplastin contains proteins, phospholipid and glycoprotein, which act as
proteolytic enzymes.

ii. The glycoprotein and phospholipid components of Thromboplastin convert

factor X into activated factor X, in the presence of factor VIII.

iii. The activated factor X reacts with factor V and phospholipid component of
tissue Thromboplastin to form Prothrombin activator (in the presence of
Calcium ions).

STAGE 2: Conversion of Prothrombin into Thrombin

Blood clothing is all about thrombin formation. Once thrombin is formed, it

definitely leads to clot formation.

i. Prothrombin is converted into thrombin by Prothrombin activator in the

presence of calcium.

ii. Once formed, thrombin initiates the formation of further molecules of

thrombin. The initially formed thrombin activates factor V. Factor V in turn
accelerates formation of both extrinsic and intrinsic Prothrombin activator
which is converted into thrombin. This effect of thrombin is called positive
feedback effect of thrombin.
STAGE 3: Conversion of Fibrinogen into fibrin.

I. Thrombin converts fibrinogen into activated fibrinogen (fibrinogema) due

to loss of two pairs of polypeptides from each Fibrinogen molecule. The
activated Fibrinogen is called fibrin Monomer.

ii. Fibrin Monomer Polymerizes with other Monomer molecules and form
loosely arranged strands of fibrin.

iii. The loose strands later are modified into dense and tight fibrin threads by
fibrin stability factor (factor XIII) in the presence of calcium ions.
Intrinsic Pathway Extrinsic Pathway
Sage I Endothelial damage + Collagen exposure Tissue trauma + Tissue Thrombopastin

Kallikrein Glycoprotein
HMW Kinogen Phospholipid

XII XIIa Platelets VIII

XI Xia Phospholipids Xa X

IX IXa Calcium
Thrombin
+
X Xa Positive
Feedback
Prothrombin Activator

Prothrombin Thrombin

Fibrinogema Fibrinogen
Polymerization
XIII
Lose strands of fibrin Fibrin tight blood clot