Plasma proteins.

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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 hematocritis about
40-45%

3. Plasmais the clear straw coloured fluid
portion of the blood minus its cellular elements.
It constitutes about 55%of the blood volume.
Serumis plasma minus clotting factors
(fibrinogen & prothrombin ).
The defribrinated plasma is called serum

4. PLASMAcontains 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 O
2,Co
2,& N
2 .

8. plasma proteins…
-forms 7% of the solids in plasma
-their normal valves –7.4 gm%
ranges from (6.4 –8.3gm%)
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 ofsodium 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 themovement 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
, βand γfractions, 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 byheat 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 animmunoregulatory 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
diseasein 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 endopeptidasessuch 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 Fe
3+
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 glycoproteinsmade up of
light(L) andheavy(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 disulfidebonds

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 variableand
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 calledhypervariable 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 amonomericmolecule 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 2
nd
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 asecretory
componentattached to dimer
Secretory IgA is found in external secretions
such as colostrum,saliva,tearsand respiratory,
intestinal & genital tract secretions
Serum IgA exists asmonomericform( 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 apentamerconsisting 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 receptorfor 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 monomerand 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 monomericmolecule 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 orbasophils
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 45
0
–60
0
C and redissolve when the
heating is continued above 80
0
C
Multiple myeloma with Bence Jones proteins in
the urine is called “light chain disease”