The term serum is applied to the liquid medium which separates out after the blood clots (coagulates). The serum does not contain fibrinogen and other clotting factors. Thus, the main difference between plasma and serum is the presence or absence of fibrinogen.
Importance of blood
The total volume of blood in an adult is around 4.5 to 5 liters. Blood performs several diversified functions. These include respiration, excretion, acid-base maintenance, water balance, transport of metabolites, hormones and drugs, body defense and coagulation.Separation of plasma proteins
The total concentration of plasma proteins is about 6-8 g/dl. The plasma is a complex mixture of proteins, and several techniques are employed to separate them. An age-old technique is based on the use of varying concentrations of ammonium sulfate or sodium sulfate. By this method, which is known as salting out process, the plasma proteins can be separated into three groups—namely albumin, globulins and fibrinogen.Electrophoresis: This is the most commonly employed analytical technique for the separation of plasma (serum) proteins.
Paper or agar gel electrophoresis with vernol buffer (pH-8.6) separates plasma proteins into 5 distinct bands namely albumin, 1, 2, and globulins. The concentration of each one of these fractions can be estimated by a densitometer.
Abnormal electrophoretic pattern
Electrophoresis of serum proteins is conveniently used for the diagnosis of certain diseases1. Multiple myeloma: A sharp and distinct M band appears in the -globulin fraction.
2. Acute infections: 1- and 2- globulins are increased.
3. Nephrotic syndrome: Decreased albumin with sharp and prominent 2-globulin.
4. Primary immune deficiency: Diminished - globulin band.
5. 1-Antitrypsin deficiency: Diminished 1- globulin band.
Albumin/globulin (A/G) ratio : The albumin concentration of plasma is 3.5 to 5.0 g/dl while that of total globulins is 2.5 to 3.5 g/dl. The normal A/G ratio is 1.2 to 1.5 : 1. The A/G ratio is lowered either due to decrease in albumin or increase in globulins, as found in the following conditions
1. Decreased synthesis of albumin by liver— usually found in liver diseases and severe protein malnutrition.
2. Excretion of albumin into urine in kidney damage.
3. Increased production of globulins associated with chronic infections, multiple myelomas etc.
ALBUMIN
Albumin is the major constituent (60%) of plasma proteins with a concentration of 3.5–5.0 g/dl. Human albumin has a molecular weight of 69,000, and consists of a single polypeptide chain of 585 amino acids with 17 disulfide bonds.Synthesis of albumin
Albumin is exclusively synthesized by the liver. For this reason, measurement of serum albumin concentration is conveniently used to assess liver function (synthesis decreased in liver diseases). Liver produces about 12 g albumin per day which represents 25% of the total hepatic protein synthesis. Albumin has an half-life of 20 days.GLOBULINS
Globulins constitute several proteins that are separated into four distinct bands (, , and -globulins) on electrophoresis.Globulins, in general, are bigger in size than albumin.
1-Antitrypsin
1-Antitrypsin, more recently called as -anti- proteinase, is a glycoprotein with 394 amino acids and a molecular weight of 54,000. It is a major constituent of 1-globulin fraction of plasma proteins with a normal concentration of about 200 mg/dl. 1-Antitrypsin is a serine protease inhibitor. It combines with trypsin, elastase and other protease enzymes and inhibits their activity.2-Macroglobulin
2-Macroglobulin concentration in plasma is elevated in nephrotic syndrome. This is due to the fact that majority of the low molecular weight proteins are lost in urine (proteinuria) in this disorder.IMMUNOGLOBULINS
The higher vertebrates, including man, have evolved a defense system to protect themselves against the invasion of foreign substances—a virus, a bacterium or a protein. The defense strategies of the body are collectively referred to as immunity.Immunoglobulins—basic concepts
Immunoglobulins, a specialised group of proteins are mostly associated with -globulin fraction (on electrophoresis) of plasma proteins. Some immunoglobulins however, separate along with and -globulins. Therefore, it should be noted that -globulin and immunoglobulin are not synonymous. Immunoglobulin is a functional term while -globulin is a physical term.Structure of immunoglobulins
All the immunoglobulin (Ig) molecules basically consist of two identical heavy (H) chains (mol. wt. 53,000 to 75,000 each) and two identical light (L) chains (mol. wt. 23,000 each) held together by disulfide linkages and non- covalent interactions Thus, immuno- globulin is a Y-shaped tetramer (H2L2). Each heavy chain contains approximately 450 amino acids while each light chain has 212 amino acids. The heavy chains of Ig are linked to carbohydrates, hence immunoglobulins are glycoproteins.Constant and variable regions: Each chain (L or H) of Ig has two regions (domains), namely the constant and the variable. The amino terminal half of the light chain is the variable region (VL) while the carboxy terminal half is the constant region (CL). As regards heavy chain, approximately one-quarter of the amino terminal region is variable (VH) while the remaining three- quarters is constant (CH1, CH2, CH3). The amino acid sequence (with its tertiary structure) of variable regions of light and heavy chains is responsible for the specific binding of immunoglobulin (antibody) with antigen.
Proteolytic cleavage of Ig: An immuno- globulin can be split by the enzyme papain to their fragments. These are two identical antigen binding fragments (Fab) and one crystallizable fragment (Fc). Papain cleaves the immunoglobin molecule at the site between CH1 and CH2 regions which is referred to as hinge region.