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1. M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

2.  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%

3.  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

4.  PLASMA contains the following composition:
WATER:
Is the main constituent of Plasma – 91%
SOLIDS: 9% of the plasma (1% inorganic molecules
& 8% organic molecules)

5. OTHER ORGANIC MOLECULES
Carbohydrates : Glucose ( 100-120 mg%)
Fats : neutral fats, phospholipids (150-
300mg%)
Cholesterol (150-240 mg%)

6.  Non protein nitrogenous substances :
ammonia, amino acids, creatine, creatinine (0.6-
1.2 mg%) xanthine, hypoxanthine, urea (20-40
mg%) & uric acid (2-4 mg%).
 Hormones enzymes & antibodies.

7.  Inorganic molecules are sodium, potassium,
calcium, magnesium, chloride, iodide, iron,
phosphates & copper.
 Gases presents in the plasma are O2 ,Co2,& N2 .

8. 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

9.  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)

10.  The albumin : globulin ratio is usually between 1.2
:; 1 to 1.5 :1
 Almost all plasma proteins , except
immunoglobulins are synthesized in liver

11.  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

12.  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.

13.  OTHER METHODS:
 Lowry’s method
 Kjeldahl’s method
 Dye-binding method
 UV-absorption method

14.  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

15.  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

16.  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.

18.  Various abnormalities can be identified in the
electrophoretic pattern
 1) CHRONIC INFECTIONS:
 The gammaglobulins are increased

19.  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

20.  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

21.  CIRRHOSIS OF LIVER :
 Albumin synthesis by liver is decreased , with
a complementary excess synthesis by globulins
by reticuloendothelial system

22.  CHRONIC LYMPHATIC LEUKEMIA:
 Gamma globulin fraction is reduced
 ALPHA-1-ANTITRYPSIN DEFICIENCY:
 The alpha-1 band is thin or even missing

23.  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.

24.  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.

25.  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.

26.  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

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

28.  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)

29. 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.

30. 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.

31. 1)BLOOD-BRAIN BARRIER:
 Albumin-fatty acid complex cannot cross blood-
brain barrier and hence fatty acids cannot be taken
up by brain.

32. 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.

33.  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.

34.  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.

35.  PROTEIN LOSING ENTEROPATHY:
 Large quantities of albumin is lost from
intestinal tract.

37.  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.

38.  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

39.  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.

40.  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

41.  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

42.  Globulins are bigger in size than albumin .
Globulins constitute several fractions. These are:
 α1- globulin
 α2- globulin
 β- globulin
 γ- globulin

43.  Retinol binding protein(RBP)
 α1 – fetoprotein(AFP)
 α1 – protease inhibitor (API)
 α1 - acid glycoprotein (AAG)
 High density lipopprotein (HDL)
 Prothrombin

44.  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.

45.  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.

46.  PROTHROMBIN
 It is synthesized by liver with the help of
vitamin K and involved in blood clotting

47.  Ceruloplasmin(ferro-oxidase)
 Transcortin / corticosteroid binding globulin
 Haptoglobin
 Thyroxine binding globulin(TBG)
 α2 - macroglobulin (AMG)

48.  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.

49.  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.

50.  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.

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

52.  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

53.  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.


54. 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

55.  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”

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

57. 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

58.  They constitute about 20% of all the plasma
proteins
 Igs are produced by plasma cells & to some extent
by lymphocytes

59.  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

60.  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)

61. Five Classes of Immunoglobulin

63.  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

64.  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)

66.  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

67.  In addition to these functions, antibodies can
act as an enzyme to catalyze the synthesis of
ozone (O3) that has microbicidal activity.

68.  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

69.  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

72.  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

73.  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 )

74.  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

77.  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

78.  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)

79.  Waldenstorm’s macroglobulinaemia :
 It is a malignant disease of lymphoid elements,
characterized by high serum concentrarion of
IgM

81.  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

83.  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

84.  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

85.  A malignant proliferation of plasma cells
 Results in an abnormally high concentration of
serum immunoglobulins, usually IgG or IgA

86.  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

87.  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”