Serum Protein: Introduction, Different Methods to Estimate, Principles, and Procedure. Reference Value, Clinical Importance,

SERUM PROTEIN

Types of proteins: Plasma proteins are a complex mixture of simple proteins. Simple proteins in plasma are albumin, globulin, fibrinogen, glycoproteins, lipoproteins, hormones, etc. Plasma proteins are synthesized inside liver cells except immunoglobulins and hormones. Fibrinogen is absent in serum. Thus serum contains all proteins except fibrinogen.

Functions/Importance of plasma/serum proteins:

1. Transportation of various molecules in blood

2. Maintenance of blood osmotic pressure & water exchange.

3. Maintenance of blood pH, and blood buffer system.

4. Tissue repairs and tissue growth

5. Immunity.

6. Coagulation of blood.

7. Body protein reserve.

Method to estimate proteins in serum:

1. Total serum proteins estimation: Total serum proteins are estimated by the Biuret method.

Principle: Cupric ions react with peptide bonds of protein molecules in alkaline media to produce violet or purple-colored copper complexes. The intensity of color is directly proportional to the concentration of protein in the specimen. The intensity of color is measured by a photometer at 530nm (green filter).

Reagents:

i. Biuret reagent (stock solution): sodium potassium tartrate 45 gm solution in 400 ml of 0.2N sodium hydroxide. Add copper sulfate 15 gm and potassium iodide 5.0gm. Add 0.2N Sodium hydroxide to make a volume up to 1000ml.

ii. Working Biuret reagent (working solution): Stock solution 200ml and 800ml of 0.2N Sodium hydroxide containing potassium iodide 5gm/L.

iii. Standard protein: 6.0gm/dl containing 0.1gm/dl sodium azide.

Calculation: Serum protein g/dl = O.D. Test/O.D. Std X 6

Normal Range: Serum protein 6 to 8gm/dl

Clinical Significance (Clinical importance): Liver cirrhosis, other liver disease, malnutrition, etc are the main reasons for to decrease in total serum protein levels. A decrease in the level of total protein is termed hypoproteinaemia. Multiple myeloma and conditions related to globulins cause an increase in serum protein levels.

2. Serum albumin estimation

Method: Bromocresol green method

Principle: Serum albumin combines with bromocresol green in an acidic medium (pH 4.1) to develop a green-colored complex. The intensity of color is proportional to the concentration of albumin in the serum specimen. The intensity of color is measured by a photometer at 640nm (red filter).

Reagents:-

i. Albumin reagent: Dissolve succinic acid 8.85gm, bromocresol green 0.108gn, sodium azide 0.1gm, brij 4.0 ml in 900ml of distilled water. Adjust pH 4.1 by adding 1n sodium hydroxide. Add distilled water to make a volume up to 1000ml.

Albumin standard: 4.0 gm bovine albumin in Normal saline 100ml.

ii. Blank reagent: Same as albumin reagent without bromocresol green.

Procedure: Photomtery at 640nm.

Calculations:

• Serum albumin, gm/dl =O.D test/ O.D Standard X 4.

Normal Range: Serum albumin 3.2 to 4.5gm/dl

3. Serum globulin estimation:

Subtraction of serum albumin concentration from total serum protein concentration gives the value of serum globulin concentration.

Calculation: 

Serum globulin gm/dL = Total Proteins gram per dL - Albumin gm per dL

Normal Range:

• Serum globulin 2.3 to 3.5gm/dL 

• A/G ratio (albumin-to-globulin ratio): 1.2:1 to 2:1

Notes on A: G Ratio: The A: G ratio (albumin-to-globulin ratio) compares the levels of albumin and globulin in blood.

• · Albumin: Produced primarily by the liver. It is a major protein in the blood that transports hormones, vitamins, and other substances throughout the body.

• · Globulins: A group of proteins produced by both the liver and the immune system. Globulins fight infections and transport certain substances in the blood.

• · Low A: G ratio: This could indicate liver problems, inflammation, malnutrition, or certain chronic diseases.

• High A: G ratio: While less common, a high A:G ratio might be associated with dehydration or some gastrointestinal issues

Dr Pramila Singh