Chapter 23:

Plasma Proteins

Textbook of
BIOCHEMISTRY
for Medical Students
By DM Vasudevan, et
al.
TENTH EDITION
Specific Learning Objectives
The learner will be able to:
•Enumerate the different plasma proteins and their functions
•Describe the methods of estimation and fractionation by electrophoresis
•Estimate total proteins and albumin, calculate A/G ratio
•Explain the functional importance of albumin, causes of hypoalbuminemia
•List the different transport proteins and their alterations in diseases
•Enumerate the acute phase reactant proteins and their relevance in clinical
practice
•Identify the different fractions in serum protein electrophoresis and detect
the presence of monoclonal spike
•Outline the coagulation cascade
•Mention the major abnormalities in coagulation process and list the tests of
coagulation
Competencies covered

BI 11.8 Demonstrate estimation of serum proteins,

albumin and A:G ratio

BI 11.21 Demonstrate estimation of glucose, creatinine,

urea and total protein in serum.

BI 11.22 Calculate albumin: globulin (AG) ratio and

creatinine clearance
Plasma Proteins
Total Protein : 6 - 8 g / 100 ml

A L B U M I N : 3.5 - 5 g /100 ml

GLOBULIN : 2.5 - 3.5 g/100ml

Electrophoresis
Electrophoresis
Movement of charged particles when subjected to an
electric field.
Positively charged particles (cations) move to cathode and
negatively charged ones (anions) to anode.
Proteins exist as charged particles.
Widely used for separation of proteins in biological fluids.
Factors Affecting Electrophoresis
The rate of migration (separation of particles) during electrophoresis
will depend upon :
1. Net charge on the particles (pI of proteins)
2. The pH of the medium
3. Mass and shape of the particles.
4. Strength of electrical field.
5. Properties of the supporting medium
6. Temperature.
Support Medium for Electrophoresis
Filter paper electro-phoresis for 16–18 hours at a low voltage.
Disadvantages
Long time
Diffusion of particles leading to blurring of margins are the of paper.
Cellulose acetate membrane strips.
Only one hour
Separation without diffusion.
identification of lipoproteins,
isoenzymes and hemoglobins.
Agarose Gels
heterogeneous polysaccharides viscous liquid when hot but
solidify to a gel on cooling.
The gel is prepared in the buffer and spread over slides and allowed
to cool. A small sample (few microlitres) of serum or biological
fluid is applied.
Electrophoretic run takes 90 minutes.
Serum proteins
protein mixtures
nucleic acids.
Visualisation of Protein Bands
After the run, the proteins are fixed to the solid support using
acetone or methanol. Then stained by (Amido Schwarz,
Ponceau S or Coomassie Blue).
The Electrophoretogram Scanned Using a Densitometer
Light is passed through agar gel plate; absorption of light
will be proportional to the quantity of protein
present on a band.

Normal and abnormal electrophoretic patterns.

1 = Normal pattern;
2 = Multiple myeloma
(M-band) between beta and gamma region.
1 = Normal pattern;
3 = Chronic infection, broad based increase in gamma region;
general increase in alpha1 and alpha2 bands;
1 = Normal pattern;
4 = Nephrotic syndrome; hypoalbuminemia; prominent
alpha-2 band
1 = Normal pattern;
5 = Cirrhosis of liver; decreased albumin band
1 = Normal pattern;
6 = Plasma showing fibrinogen (normal condition) which may
be mistaken for paraproteins.
Serum electrophoretic pattern, as seen in agar gel. Lanes 2, 4, 8, 10 = Normal
pattern. Lane 1 = Nephrotic syndrome, hypoalbuminemia, prominent alpha-2
band. Lane 3 = Cirrhoris, hypoalbuminemia with beta-gamma bridging. Lane
5 = Chronic infection, broad based increase in gamma region, general increase
in alpha-1 and alpha-2 bands, comparative reduction albumin band. Lanes 6, 7
= Multiple myeloma, monoclonal band (M band) between beta and gamma.
Lane 9 = Acute inflammation, reduced albumin and increased alpha-2.
Albumin
Latin, albus = white
Mol. Wt. 69,000
Synthesised by hepatocytes 25% of total hepatic protein synthesis
Important Liver Function Test
Functions:
1. Effective Osmotic Pressure
Colloidal Osmotic Pressure
2. Transport of hydrophobic substances
3. Buffering (histidine residues)
4. Nutritional
Total Osmotic Pressure : 5000 mm Hg
Effective Osmotic pressure: 25 mmHg
80% by albumin; 20% globulins
Edema when Albumin < 2 g / dl
1) Albumin is reduced
A) Malnutrition; generalised edema synthesis is reduced
B) Nephrotic syndrome; facial edema; Loss of albumin
C) Cirrhosis; Ascites; synthesis Albumin is a Liver Function
Test Half life is about 20 days
2) Congestive Cardiac Failure
Dependent edema, feet Increased hydrostatic P in vein
Functions of Albumin
1. Effective Osmotic Pressure
[Link] of hydrophobic substances

Specific carriage
Bilirubin
Non-esterified fatty acids (NEFA)

Non-specific carriage
Steroids, Thyroxin, Calcium, Copper Aspirin, Sulpha drugs
Dicoumarol, Phenytoin
Albumin-fatty acid complex cannot cross blood brain barrier
Bilirubin- aspirin interaction Drug interactions
Kernicterus mental retardation
Biological availability of drugs
Protein bound calcium Hypoalbuminemia Decreased calcium in
blood
Normal Albumin level in blood:
3.5 - 5 g /dl
Hypoalbuminemia
Cirrhosis, liver failure
Malnutrition
Malabsorption syndromes
Nephrotic syndrome
Proteinuria - albuminuria
Heat and acetic acid test
Proteinuria
Glomerular proteinuria
Micro albuminuria
Minimal albuminuria
Pauci albuminuria
Small quantities of albumin Less than 300 mg per day
Diabetes mellitus
Hypertension
Indicator of future renal failure
Hypoproteinemia
Cirrhosis
Nephrotic syndrome
Malnutrition
Malabsorption syndromes
Reversal of Albumin : Globulin
Reduction of albumin Compensatory increase in globulin fractions
Hyper Globulinemias
Reduction of albumin
Chronic infections (gamma)
Multiple myeloma (gamma)
Lipoproteinemias (beta) Atherosclerosis
Nephrotic syndrome (alpha)
Normal Values
Albumin : 55-65%

Alpha-1-globulin : 2- 4%

Alpha-2-globulin : 6-12%

Beta-globulin : 8-12%

Gamma-globulin : 12-22%
Globulins
• Globulins are Precipitated by Half Saturation with Salts
• [Link] 90,000 - 1,30,000

• α1 GLOBULIN
Synthesized in Liver
• α2 GLOBULIN

  GLOBULIN
Reticulo Endothelial System
  Globulin
α1 Globulin
• Complex Proteins:0.42 g/dl
• α1 Acid Glycoprotein

• α1 Lipoprotein

• α1 fetoprotein

• α1 Globulin Inhibitors

• α1 Antitrypsin
α2 Globulin
• Complex Proteins:0.67 g/dl
• α2 Glycoprotein
• Caeruloplasmin
• Haptoglobin
• Plasminogen
• Prothrombin
• α2 Macroglobulin
 Globulin
• 0.91 d/dl
• β Lipoprotein-LDL
• Transferrin
• C Reactive Proteins
• Hemopexin
• Complement Proteins
• β Micro Globulin
 Globulin
• These are Mainly Immunoglobulins
Fibrinogen
• Soluble Glycoprotein
• 4-6% Plasma Protein
• 200 - 400 mg/dl
• M Wt 3,50,000 - 4,50,000
• Made up of 6 Polypeptide Chains
• Asymmetrical and Elongated Molecules
Fibrinogen
• Synthesized in Liver
• Decreased in Liver Disease
• Precursor of Fibrin
Transport Proteins
for carrying lipid substances
1. ALBUMIN
Bilirubin, fatty acids, Calcium, drugs
2. TRANSTHYRETIN (TBPA)
Thyroxin binding pre-albumin
3. Thyroxin binding globulin (TBG)
 Pregnancy;  Nephrotic
4. Retinol binding protein (RBP) Half life, 2-3 days
Transport Proteins
5. Transcortin
Cortisol binding globulin (CBG)
 Pregnancy
6. Haptoglobin (for Hb)
7. Hemopexin (for Heme)
8. Transferrin (for Iron)
9. HDL (High Density Lipoproteins) (Cholesterol)
10. LDL (Low Density Lipoproteins) (Cholesterol)
Carrier Proteins or Transport Proteins of Plasma

Name Plasma level Compound bound or Biological and clinical

transported significance

Albumin 3.5–5 g/dl Fatty acids, bilirubin, Bilirubin competes with

calcium, thyroxine, aspirin for binding sites on
heavy metals, drugs albumin

Pre-albumin (Trans- 25–30 mg/dl Steroid hormones, Rich in tryptophan. Half-

thyretin) Thyroxine, Retinol life is 1day. It is a negative
acute phase protein.
Transports T3 and T4
losely.

Retinol binding 3–6 mg/dl Retinol Synthesised by liver. RBP

protein (RBP) (Vitamin A) has a short half-life. Level
indicates vitamin A status.
Useful to assess the
protein turn over.
Carrier Proteins or Transport Proteins of Plasma

Name Plasma level Compound bound Biological and clinical

or transported significance

Thyroxine binding 1–2 mg/dl Thyroxine Assessment of the binding

globulin sites on TBG is important in
(TBG) studying thyroid function.
It is synthesised in liver

Trans-cortin; 3–3.5 mg/dl Cortisol and Cortico- Synthesised by liver.

Cortisol Binding sterone Increased in pregnancy. Free
globulin unbound fraction of hormone
is biologically active.

Hapto-globin (Hp) 40-175mg/dl Hemoglobin Synthesised in liver. Low

level indicates hemolysis.
Half-life of Hp is 5 days; but
that of Hb-Hp is only 90
minutes. It is an acute phase
protein
Carrier Proteins or Transport Proteins of Plasma

Name Plasma level Compound bound or Biological and clinical

transported significance

Transferrin 200–300 mg/dl Iron 33% saturated Conserves iron by preventing

iron loss through urine

Hemopexin 50–100 mg/dl Free heme Helps in preventing loss of

heme (and so iron also) from
body.
HDL (High Cholesterol The lipoprotein contains
density lipo- Phospho-lipids apoprotein-A.
protein) Serves to transport cholesterol
from tissues to liver for
elimination through bile. It is
anti-atherogenic.

LDL (Low density Cholesterol; Phospho- Contains apoprotein-B. Trans-

lipo-protein) lipids; TG ports cholesterol to tissues. It
increases risk for MI.
Acute Phase Proteins
50-1000 times
Inflammatory / neoplastic diseases
Induction by Interleukins released by macro / lymphocytes
C-reactive protein
Ceruloplasmin
Haptoglobin
Alpha-1 anti trypsin
Fibrinogen
C-reactive Protein (CRP)
C-polysaccharide of capsule of pneumococci Beta globulin;
Synthesised in liver
CRP+Bacteria -(similar to antibodies) Complement activation
Bacteria killed CRP level parallels with
inflammation, trauma
Follow up of patients
High sensitivity CRP (hs-CRP)
Predictive value for
Myocardial infarction
Ceruloplasmin
Latin, blue
Alpha-2 globulin
Mol wt. 160,000
Synthesised in liver
6-8 copper atoms per molecule
FERROXIDASE
Ceruloplasmin is an enzyme antioxidant in plasma
Cp is not a transport protein
Albumin is carrier for Copper
Ceruloplasmin
Normal blood level: 25-50 mg /dl
Reduced in Wilson’s disease
Hepato lenticular degenartion
Inherited autosomal recessive
Copper is not excreted
Copper toxicity
 copper into ceruloplasmin
Copper Deposited in Organs
Hepatic degeneration Cirrhosis
Basal ganglia;
Lenticular degeneration
Neurological symptoms
Kidney - Renal failure
Bone marrow: Hemolytic anemia
Treatment: Low copper
Low Cp: Malnutrition
Nephrotic syndrome
Cirrhosis
Increase Cp: Inflammation
Alpha-1 Antitrypsin (AAT)
Alpha anti proteinase
Protease inhibitor (Pi)
inhibits plasmin, thrombin, cathepsin, trypsin, chymotrypsin, elastase
Serine Protease Inhibitors
SERPINs
Synthesised in liver
Bulk of alpha-1 mobility
Multiple allelelic forms
75 variant forms
Polymorphism
Protein exists in different phenotypes in the population
( > 1%) ; but only one form is seen in a particular person
ISOFORMS
Variants in molecules seen in all normal persons, eg, Classes of
immunoglobulins
MUTATION
Only in a few individuals (<1%)
AAT Deficiency
Alpha-1 band absent
1 in 1000; most common inborn error
Emphysema
Lung infection macrophage Elastase Tissue
destruction Normally this is opposed by AAT In the deficient
person
continued action of elastase destruction of lung Emphysema
About 5% cases of emphysema are due to AAT deficiency
Smoking will inactivate AAT So, in smokers, emphysema is
common
Alpha-2 Macroglobulin (AMG)
[Link]. 725,000
Major component of alpha-2 proteins
Hepatocytes and macrophages

AMG inactivates all proteases in vivo anti-coagulant

Increased in Nephrotic syndrome

Negative Acute Phase Proteins
Transthyretin (Pre-albumin)

Half life of 7-10 days

Hence a better index for protein turn over than albumin
C-Reactive Protein (CRP)
C-polysaccharide of capsule of pneumococci
Beta globulin; Synthesised in liver
CRP + Bacteria = Complement
activation = Bacteria killed
CRP level parallels with
inflammation, trauma
Follow up of patients

High sensitivity CRP (hs-CRP)

Predictive value for Myocardial infarction
Ceruloplasmin
Latin, blue
Alpha-2 globulin
Mol wt. 160,000
Synthesised in liver
6-8 copper atoms per molecule
FERROXIDASE
Ceruloplasmin is an enzyme antioxidant in plasma
Cp is not a transport protein
Albumin is carrier for Copper
Ceruloplasmin
Normal blood level: 25-50 mg /dl
Reduced in Wilson’s
Hepato lenticular degenartion
Inherited autosomal recessive
Mutation in gene encoding a copper binding ATPase in cells which
is required for excretion of copper
So copper is not excreted
Copper toxicity
 copper into ceruloplasmin
Copper Deposited in Organs
Hepatic degeneration Cirrhosis
Basal ganglia;
Lenticular degeneration
Neurological symptoms
Kidney Renal failure
Bone marrow: Hemolytic anemia
Treatment: Low copper
Low Cp: Malnutrition
Nephrotic syndrome
Cirrhosis
Increase Cp: Inflammation
Haptoglobin
• Glycoprotein that binds extra corpuscular Hemoglobin
• 40 -180 mg/dl
• M wt: 90,000
• Prevents the loss of free Hb through the kidney
• Synthesized in liver
• Increased in various inflammatory states
• Decreased in hemolytic anemia
Clotting Factors
The biochemical mechanism of clotting is a typical example
of cascade activation. All the coagulation factors are present
in the circulation as inactive zymogen forms. They are converted to
their active forms only when the clotting process is initiated. This
would prevent unnecessary intravascular coagulation.

Activation process leads to a cascade amplification effect, in which

one molecule of preceding factor activates so many molecules of the
next factor. Thus within seconds, a millions of molecules of final
factors are activated.
Several of these factors require calcium for their activation.
The calcium ions are chelated by the gamma carboxyl group
of glutamic acid residues of the factors, prothrombin, VII, IX, X, XI
and XII. The gamma carboxylation of glutamic acid residue is
dependent on vitamin K, and occurs after the synthesis of the protein
(post-translational modification).
Cascade Pathway of Coagulation
Factors Involved in Coagulation Process
No. Name Activated by Function

I Fibrinogen Thrombin Forms the clot (fibrin)

II Prothrombin Factor Xa Activation of fibrinogen and

factors XIII, VIII and V

IV Calcium — Activation of factor II, VII,

IX, X, XI and XII

V Labile factor Thrombin Binding of prothrombin to

platelet

VII Proconvertin; (SPCA) Thrombin Activation of factor X

VIII Antihemophilic globulin Thrombin Activation of factor X

(AHG)
Factors Involved in Coagulation Process

No. Name Activated by Function

IX Plasma thromboplastin- Factor Xla Activation of factor X

component (PTC);
Christmas factor

X Stuart Prower factor Factor IXa Activation of prothrombin

XI Plasma thromboplastin Factor XIIa Activation of factor IX
anticedent (PTA)

XII Hageman factor Kallikrein Activation of factor XI

XIII Fibrin stabilising factor Thrombin Stabilization of fibrin clot by

(Liki Lorand factor) forming cross-links
Prothrombin
It is a single chain zymogen with a molecular weight of 69,000D.
The plasma concentration is 10–15 mg/dL. The prothrombin is
converted to thrombin.
Thrombin
It is a serine protease with molecular weight of 34,000 D.
The Ca++ binding of prothrombin is essential for anchoring
the prothrombin on the surface of platelets. When the terminal
fragment is cleaved off, the calcium binding sites are removed and
so, thrombin is released from the platelet surface.
Fibrinogen
The conversion of the fibrinogen to fibrin occurs by cleaving
of Arg-Gly peptide bonds of fibrinogen. The fibrinogen has a
molecular weight of 3,40,000 D and is synthesized by the liver.
Normal fibrinogen level in blood is 200–400 mg/dL. The fibrin
monomers formed are insoluble. They align themselves lengthwise,
aggregate and precipitate to form the clot. Fibrinogen is an acute
phase protein.
Prothrombin Time
Prothrombin time (PT) evaluates the extrinsic coagulation pathway,
so that if any of the factors synthesized by the liver (factors I, II, V,
VII, IX and X) is deficient, the PT will be prolonged. PT is the time
required for the clotting of whole blood (citrated or oxalated) after
addition of calcium and tissue thromboplastin. So, fibrinogen is
polymerized to fibrin by thrombin.
Fibrinolysis
Unwanted fibrin clots are continuously dissolved in vivo by
plasmin, a serine protease. Its inactive precursor is
plasminogen (90 kD). It is cleaved into two parts to produce the
active plasmin. Plasmin, in turn, is inactivated by the alpha-2
antiplasmin.
Tissue plasminogen activator (TPA) is a serine protease present in
vascular endothelium. The TPA is released during injury and then
binds to fibrin clots. Then TPA cleaves plasminogen to generate
plasmin, which dissolves the clots.
D-Dimer
It is the protein fragment that forms in blood soon after the
degradation of a blood clot and later gets cleared by the kidney. In
Covid pandemic complications, the formation of higher level of blood
clots in blood will lead to severe fatal pneumonia, pulmonary
embolism or vein thrombosis. The venous thromboembolism can be
identified by the D-dimer test.

D-dimer is one of the natural biomarkers for the extent of blood

clotting. So, people who have a tendency for blood clotting
disorders should mandatorily do a D-dimer test to know the extent of
clot formation. The D-dimer level of above 0.5 milligrams per mL of
blood or higher values means abnormal blood clotting and needs
immediate treatment.
Clinical Significance of Clotting Factors
Thrombosis in coronary artery is the major cause of
myocardial infarction (heart attack). If the TPA, urokinase or
streptokinase is injected intravenously in the early phase of
thrombosis, the clot may be dissolved and complete recovery of
patient is possible.
Hemophilia A (Classical Hemophilia)
This is an inherited X-linked recessive disease affecting males and
transmitted by females. Male children of hemophilia patients are
not affected; but female children will be carriers, who transmit the
disease to their male offspring. This is due to the deficiency of factor
VIII (antihemophilic globulin) (AHG). It is the most common of
the inherited coagulation defects.
There will be prolongation of clotting time. Hence, even trivial
wounds such as tooth extraction will cause excessive loss of blood.
Patients are prone to internal bleeding into joints and intestinal tract.
Hemophilia A (Classical Hemophilia)
Until the recent times, the treatment consisted of administration of
AHG, prepared from pooled sera every three months. Since this was
not generally available, the standard treatment was to transfuse blood
periodically, which may lead to eventual iron overload, called
hemochromatosis. Several hemophilia patients, receiving repeated
trans-fusions became innocent victims of AIDS. Pure AHG is now
being produced by recombinant technology and is the treatment of
choice.
Inheritance Pattern of Hemophilia.
Hemophilia B or Christmas Disease
It is due to factor IX deficiency. The Christmas disease is named
after the first patient reported with this disease.
Similar deficiencies of factors X and XI are also reported. In these
diseases, the manifestations will be similar to classical hemophilia.
Other Disorders
Acquired hypofibrinogenemia or a fibrinogenemia may occur as a
complication of premature separation of placenta or abruption
placenta. Proteolytic thromboplastic substances may enter from
placenta to maternal circulation which sets off the clotting cascade
(disseminated intravascular coagulation or DIC). But the clots are
usually degraded immediately by plasminolysis. Continuation of this
process leads to removal of all available prothrombin and fibrinogen
molecules leading to profuse postpartum hemorrhage.
Anticoagulants
They are two types: 1. Acting in vitro to prevent coagulation of
collected blood, and 2. Acting in vivo to prevent and regulate
coagulation. The first group of anticoagulant removes calcium
which is essential for several steps on clotting. Oxalates, citrate and
EDTA belong to this group.
Heparin and antithrombin III are the major in vivo
anticoagulants. Heparin is also used as an anticoagulant for in vitro
system, e.g. in dialysis and for the treatment of thrombo-embolic
diseases. It is also used in the treatment of intravascular
thrombosis. Low molecular weight heparin is used to prevent DVT.
Antagonists to vitamin K are used as anticoagulants, especially for
therapeutic purposes, e.g. Dicoumarol and Warfarin.