M.Prasad NaiduMSc Medical Biochemistry, Ph.D,.

Total blood volume is 4.5-5 litres.

If blood containing anticoagulants (e.g.heparin , potassium oxalate) is centrifuged , the plasma separates out as a supernatant while the cells remain at the bottom.

About 55-60% of blood is plasma

The packed cell volume or hematocrit is about 40-45%

Plasma is the clear straw coloured fluid portion of the blood minus its cellular elements.

It constitutes about 55% of the blood volume.

Serum is plasma minus clotting factors (fibrinogen & prothrombin ).

The defribrinated plasma is called serum

PLASMA contains the following composition:

WATER: Is the main constituent of Plasma – 91%Is the main constituent of Plasma – 91%

SOLIDS: : 9% of the plasma (1% inorganic molecules 9% of the plasma (1% inorganic molecules

& 8% organic molecules)& 8% organic molecules)

OTHER ORGANIC MOLECULESOTHER ORGANIC MOLECULES

Carbohydrates : : Glucose ( 100-120 mg%)Glucose ( 100-120 mg%)

Fats : : neutral fats, phospholipids (150-300mgneutral fats, phospholipids (150-300mg%)%)

Cholesterol (150-240 mg%)(150-240 mg%)

Non protein nitrogenous substances : :

ammonia, amino acids, creatine, creatinine ammonia, amino acids, creatine, creatinine

(0.6-1.2 mg%) xanthine, hypoxanthine, urea (0.6-1.2 mg%) xanthine, hypoxanthine, urea

(20-40 mg%) & uric acid (2-4 mg%).(20-40 mg%) & uric acid (2-4 mg%).

Hormones enzymes & antibodies.Hormones enzymes & antibodies.

Inorganic molecules are sodium,

potassium, calcium, magnesium, chloride,

iodide, iron, phosphates & copper.

Gases presents in the plasma are O2 ,Co2,& N2 .

plasma proteins… - forms 7% of the solids in plasma - their normal valves – 7.4 gm%

ranges from (6.4 – 8.3 gm%)

INCLUDES: ALBUMIN

GLOBULINS FIBRINOGEN

Total protein content of normal plasma is 6 - 8 g/100ml

The plasma proteins consist of :

1)albumin (3.5-5 g/dl)

2)globulins (2.5-3.5 g/dl)

3)fibrinogen (200-400 mg/dl)

The albumin : globulin ratio is usually between 1.2 :; 1 to 1.5 :1

Almost all plasma proteins , except immunoglobulins are synthesized in liver

In clinical laboratory, separation is usually done by salts.

Thus , fibrinogen is precipitated by 10% and

globulins by 22% concentration of sodium

sulphate

Ammonium sulphate will precipitate : albumin by full-saturation globulin by half-saturation

In clinical laboratory , total proteins of patients are estimated by Biuret method.

Albumin is quantitated by Bromo cresol

green (BCG) method , in which the dye is

preferentially bound with albumin , and the

colour intensity is measured colourimetrically.

OTHER METHODS:

Lowry’s method

Kjeldahl’s method

Dye-binding method

UV-absorption method

The most common method of analyzing

plasma proteins is by electrophoresis.

The term electrophoresis refers to the

movement of chargeD particles through

an electrolyte when subjected to an

electric field

In clinical laboratory , cellulose acetate is widely used as a supporting medium.

Its use permits resolution , after staining , of plasma proteins into five bands , designated albumin , α1 , α2 , β and γ fractions, respectively

The stained strip of cellulose acetate is

called electrophoretogram.

The amounts of these five bands can be

conveniently quantified by use of

Densitometric scanning machines.

Characteristic changes in the amounts of one

or more of these five bands are found in

many diseases.

Various abnormalities can be identified in the electrophoretic pattern

1) CHRONIC INFECTIONS: The gammaglobulins are increased

2)MULTIPLE MYELOMA :

In para-proteinemias , a sharp spike is noted and is termed as M-band.

This is due to monoclonal origin of immunoglobulins

PRIMARY IMMUNE DEFICIENCY :

The gamma globulin fraction is reduced

NEPHROTIC SYNDROME :

All proteins except very big molecules are lost through urine , and α-2-fraction will be very prominent

CIRRHOSIS OF LIVER :

Albumin synthesis by liver is decreased , with a complementary excess synthesis by globulins by reticuloendothelial system

CHRONIC LYMPHATIC LEUKEMIA: Gamma globulin fraction is reduced

ALPHA-1-ANTITRYPSIN DEFICIENCY: The alpha-1 band is thin or even missing

Albumin (69 kDa) is the major protein in human plasma(3.4-4.7 g/dl)

It makes up approximately 60% of the total plasma protein.

About 40% of albumin is present in the plasma, and the other 60% is present in the extracellular space.

The liver produces about 12g of albumin per

day , representing about 25% of total hepatic

protein synthesis

Albumin can come out of vascular

compartment. So albumin is present in CSF

and interstitial fluid.

1)COLLOID OSMOTIC PRESSURE OF PLASMA:

The total osmolality of serum is 278-305

mosmol/kg.

This is exerted mainly by salts, which can pass

easily from intravascular to extravascular space.

Therefore, the osmotic pressure exerted by

electrolytes inside and outside the vascular

compartments will cancel each other.

But proteins cannot easily escape out of

blood vessels, and therefore , proteins

exert the ‘effective osmotic pressure’.

It is about 25mm Hg, and 80% of it is

contributed by albumin.

The maintenance of blood volume is

dependent on this effective osmotic

pressure

Gaw: Clinical Biochemistry; Churchill Livingstone (1999), p. 44.

TRANSPORT FUNCTION:

Albumin is the carrier of various hydrophobic substances in the blood such as:

i)bilirubin & non-esterified fatty acids ii)drugs (sulpha,aspirin,salicylate,) iii)hormones(steroid hormones,thyroxine) iv)metals (calcium,copper,heavy metals)

3)BUFFERING ACTION :

Albumin has maximum buffering capacity

amongst all proteins

It has a total of 16 histidine residues which

contribute to this buffering action.

4)NUTRITIONAL FUNCTION:

All tissue cells can take up albumin by

pinocytosis.

It is then broken down to amino acid level.

So albumin may be considered as the

transport form of essential amino acids from

liver to extrahepatic cells.

1)BLOOD-BRAIN BARRIER:

Albumin-fatty acid complex cannot cross blood-brain barrier and hence fatty acids cannot be taken up by brain.

2)PROTEIN-BOUND CALCIUM:

Calcium level in blood is lowered in hypo-

albuminemia

Thus , even though total calcium level in

blood is lowered, ionised calcium level may

be normal, so tetany may not occur.

3) THERAPEUTIC USE:

Human albumin is therapeutically useful to treat burns,hemorrhage and shock.

4)EDEMA:

Hypo-albuminemia will result in tissue edema Eg: a)malnutrition

b)nephrotic syndrome

c)cirrhosis of liver

d)chronic congestive cardiac failure.

CIRRHOSIS OF LIVER: Synthesis is decreased.

MALNUTRITION: Availability of amino acids is reduce and so

albumin synthesis is affected.

NEPHROTIC SYNDROME: Permeability of kidney glomerular

membrane is defective , so that albumin is excreted in large quantities.

PROTEIN LOSING ENTEROPATHY:

Large quantities of albumin is lost from

intestinal tract.

ALBUMINURIA:

Presence of albumin in urine is called

albuminuria.

It is always pathological.

Seen in:

a)Nephrotic syndrome(large quantities)

b)Acute nephritis

c)Inflammatory conditions of urinary tract.

Detection of albumin in urine is done by heat

and acetic acid test.

MICRO-ALBUMINURIA:

In micro-albuminuria or minimal albuminuria

or plauci-albuminuria , small quantity of

albumin (30-300 mg/dl) is seen in urine

It is estimated by RIA

Increased levels of microalbuminuria is an

indication of early involvement of renal

tissue in diabetic patients

Albumin-globulin ratio :

In hypo-albuminemia, there will be a

compensatory increase in globulins which are

synthesized by the reticulo-endothelial

system(plasma cells).

Albumin-globulin ratio (A/G ratio) is thus

altered or even reversed.

Hypoproteinemia :

Since albumin is the major protein

present in the blood, any condition

causing lowering of albumin will lead to

reduce total proteins in blood

HYPERALBUMINEMIA :

Increased levels of plasma albumin are present only in acute dehydration and have no clinical significance

ANALBUMINAEMIA :

Analbuminemia is a rare hereditary

abnormality in which plasma albumin

concentration is usually less than 1.0gm/L

Globulins are bigger in size than albumin . Globulins constitute several fractions. These are:

α1- globulin α2- globulin β- globulin γ- globulin

Retinol binding protein(RBP)

α1 – fetoprotein(AFP)

α1 – protease inhibitor (API)

α1 - acid glycoprotein (AAG)

High density lipopprotein (HDL)

Prothrombin

RETINOL BINDING PROTEIN (RBP) Retinol (vitamin A) is transported in plasma

bound to RBP. Most retinol RBP in the plasma is

reversibely complexed with transthyretin (thyroxine binding protein)

α1- FETOPROTEIN (AFP) This is present in the tissues and plasma of

the fetus It may play an immunoregulatory role

during pregnancy.

α1- PROTEASE INHIBITOR (API) / α1- ANTITRYPSIN (AAT) :

API is one of the plasma proteins, that inhibits

activity of proteases particularly elastase, which

degrades elastin, a protein that gives elasticity to

the lungs

α1-ACID GLYCOPROTEIN (AAG)

AAG also known as orosomucoid, contains a high

percentage of carbohydrate with a large number

of sialic acid residues

It is synthesized by liver parenchymal cells.

PROTHROMBIN

It is synthesized by liver with the help of

vitamin K and involved in blood clotting

Ceruloplasmin(ferro-oxidase)

Transcortin / corticosteroid binding

globulin

Haptoglobin

Thyroxine binding globulin(TBG)

α2 - macroglobulin (AMG)

CERULOPLASMIN (FERRO-OXIDASE)

This is a copper containing protein.

It has oxidase activity

Ceruloplasmin is the major transport protein for

copper, an essential trace element.

It is also essential for the regulation of oxiation-

reduction , transport and utilization of iron

Plasma ceruloplasmin level is reduced in

Wilson’s disease in patients with malnutrition

and in the nephrotic syndrome.

TRANSCORTIN /CORTICOSTEROID BINDING GLOBULIN:

This binds cortisol It is synthesized in liver and synthesis is increased

by oestrogen

HAPTOGLOBIN:

It plays an important role in the conservation of iron by preventing its loss in the urine

Haptoglobin binds free Hb to form a complex which is too large to be filtered by the kidney and thus prevents the loss of iron in the urine.

THYROXINE-BINDING GLOBULIN (TBG)

TBG is synthesized in liver TBG has a electrophoretic mobility between α1 & α2 globulins

It transports thyroxine hormone(T3 & T4)

α2 - MACROGLOBULIN(AMG)

This is major α2 - globulin , which is a

natural inhibitor of endopeptidases such

as trypsin, chymotrypsin, plasmin,

thrombin .etc.

Haemopexin Transferrin β2 -microglobulin(BMG) C-reactive protein(CRP) Low density lipoprotein

HAEMOPEXIN

Like haptoglobulin, haemopexin also plays

an important role in the conservation of iron

by preventing its loss in urine

TRANSFERRIN

Is synthesized in liver

It transports iron(2 molecules of Fe3+ per

molecule of transferrin) through blood to the

sites where iron is required

C-REACTIVE PROTEIN(CRP)

CRP is involved in the body’s response to inflammations .mainly bacterial..

It is useful in differentiating bacterial from viral infections because the level of CRP is increased in bacterial infections only.

MICROGLOBULIN

This protein forms part of the human

leucocyte antigen(HLA) system

Plasma levels are increased whenever, there

is malignant lymphoid or myeloid

proliferation and renal failure

The acute phase response is a non-specific response to the stimulus of tissue following trauma, infection ,inflammation, burn, etc

Following trauma etc , the body responds by initiating a series of mechanisms that lead to rapid decrease in the concentration of many proteins,eg

Albumin Prealbumin Transferrin

These are termed “negative acute phase reactants”

An increase in the concentration of several specific proteins occur some hours after the injury. These proteins are called the positive acute phase proteins

Definition:

The Igs constitute a heterogenous family of serum proteins, which either function as antibodies or are chemically related to antibodies

The immunoglobulins are γ- globulins , called antibodies. All antibodies are immunoglobulin but all immunoglobulins may not be antibodies

They constitute about 20% of all the plasma proteins

Igs are produced by plasma cells & to some extent by lymphocytes

Immunoglobulins are glycoproteins made up of light(L) and heavy(H) polypeptide chains.

All Igs have the same basic structure.

The basic Ig is a “Y” shaped molecule and consist of 4 polypeptide chains:

2 H chains 2 L chains

The 4 chains are linked by disulfide bonds

An individual antibody molecule always consists of identical H chains & identical L chains

L chain may be either of 2 types, kappa(κ) or lambda(λ) but not both

The heavy chains may be of 5 types and are designated by greek letter:

Alpha(α) Gamma(γ) Delta(δ) Mu(μ) Epsilon(ε)

Five Classes of Immunoglobulin

Igs are named as per their heavy chain type as IgA , IgG , IgD , IgM & IgE

The L and H chains are subdivided into variable and constant regions

L chain consists of one variable(VL) and one constant (CL) domain or region

Most H-chains consist of one variable(VH) and 3 constant(CH-1,CH-2 & CH-3) domains

IgG & IgA have 3 CH domains whereas IgM & IgE have 4

Each Ig molecule has hinge region between CH-1 & CH-2, which allows better fit with the antigen surface.

The variable regions of both L & H chains have 3 extremely variable amino acid sequences at the amino terminal end called hypervariable region

Enzyme(papain) digestion splits the Ig molecule into 2 fragments named as Fab (Fragment for antigen binding) and Fc (crystallizable fragment)

The primary function of antibodies is to protect against infectious agents or their products.

Igs provide resistance because they can :

Neutralize toxins & viruses Opsonize microbes so they are more easily

phagocytosed Activate complement & prevent the

attachment of microbes to mucosal surfaces

In addition to these functions, antibodies

can act as an enzyme to catalyze the

synthesis of ozone (O3) that has

microbicidal activity.

IgG (HEAVY CHAIN γ ) :

Is a monomeric molecule with 2 antigen binding sites

There are 4 subclasses, IgG1 to IGg4 based on antigenic differences in the H-chains and on the number and location of disulfide bonds

It is produced mainly in the secondary response and constitutes an important defence against bacteria & viruses

IgG is the major class of immunoglobulin found in the serum which accounts for 70% of the total

IgG is the only antibody that crosses the placenta & therefore is the class of maternal antibody that protects the fetus

Functions: Neutralizes bacterial toxins and viruses Opsonises bacteria, making them easier to

phagocytize Activates complements which enhances

bacterial killing

IgA is the 2nd most abundant class constituting about 20% of serum immunoglobulins

IgA occurs in 2 forms:

Secretory IgA Serum IgA

Secretory IgA is a dimeric molecule formed

by 2 monomer units, joined together at their

carboxy terminals by a protein termed J-chains

Additionally secretory IgA has a secretory component attached to dimer

Secretory IgA is found in external secretions such as colostrum,saliva,tears and respiratory , intestinal & genital tract secretions

Serum IgA exists as monomeric form( found in internal secretions such as synovial,amniotic,pleural & CSF )

Functions:

Secretory IgA prevents attachment of

bacteria and viruses to mucous

membranes and helps protect mucous

surface from antigenic attack

Prevents access of foreign substances to

circulation

It is a pentamer consisting of 5 identical Ig molecules, joined together by disulfide bridges.

IgM accounts for some 10% of normal Ig

IgM is the main Ig produced early in the primary response

As it is pentamer, it has 10 antigen binding sites & is the most efficient Ig in agglutination, complement activation & other antibody reactions & is important in defence against bacteria & viruses

The natural blood group antibodies, anti-A & anti-B are IgM

IgM present on the surface of B lymphocytes is monomer, where it functions as an antigen binding receptor for antigen recognition

IgM can be produced by fetus in certain infections.

Functions:

Activate complement, promotes phagocytosis & causes lysis of antigenic cells(bacteria)

Waldenstorm’s macroglobulinaemia :

It is a malignant disease of lymphoid elements, characterized by high serum concentrarion of IgM

It is a monomer and resembles IgG

structurally

IgD has no known antibody function but may

function as an antigen receptor

Like, IgM, it is present on the surface of many B

lymphocytes

The circulating concentration of IgD in blood is

very low

IgD is labile

IgE is a monomeric molecule similar to IgG. It is sometimes called reagin

Although IgE is present in trace amounts, in normal persons with allergic activity have greatly increased amounts

Functions:

Antiallergic & antiparasitic

IgE is responsible for anaphylactic(immediate) type of hypersensitivity & allergy. Its main activity is mediated by mast cells or basophils

Defends against worm infections by causing release of enzymes from eosinophils

Main host defence against parasites like helminthus, provides protection in the disease schistomiasis

A malignant proliferation of plasma cells

Results in an abnormally high concentration of serum immunoglobulins, usually IgG or IgA

In multiple myeloma, more light chains are produced than heavy chains and enter the bloodstream

Because they are of relatively low m.wt, they pass through glomerular membrane and appear in the urine, these protein chains of low m.wt are known as Bence Jones Proteins

Bence Jones proteins have the

remarkable characteristic of precipitating

on heating urine from 450 – 600 C and

redissolve when the heating is continued

above 800 C

Multiple myeloma with Bence Jones

proteins in the urine is called “light

chain disease”