Estimation of Total Protein by kit method

Introduction:
Plasma proteins are derived primarily from synthesis in the liver, plasma cells, lymph nodes, spleen, and
bone marrow. In disease states both the total plasma protein level and the ratio of the individual
fractions may be dramatically altered from their normal values. Hypoproteinemia may be caused by such
conditions as nephrotic syndrome, extensive bleeding, sprue (deficient protein absorption), severe
burns, salt retention syndromes, cirrhosis, kidney damage and Kwashiorkor (acute protein starvation).
Hyperproteinemia may be observed in cases of severe dehydration and disease states such as multiple
myeloma. Changes in the proportions of the plasma proteins may occur in one or several of the protein
fractions and often without alterations in the quantity of the total protein. The A/G ratio has commonly
been used as an index of the distribution between the albumin and globulin fractions. This ratio can be
significantly altered in such conditions as cirrhosis of the liver, glomerulonephritis, nephrotic syndrome,
acute hepatitis, lupus erythematosis, and in some acute and chronic infections.

Principle: This test is performed using the Biuret Method.

The peptide bonds (-CO-NH) present in the protein react with copper sulfate in an alkaline medium to
form a purple/violet colored complex. Polypeptides containing at least two peptide bonds react with
biuret reagent. Cupric ion forms a coordination complex with protein nitrogen with very little difference
between albumin and globulin. The intensity of this color is proportional to the number of peptide
linkages present and thus is a measure of the concentration of proteins.

Reagents:

 Biuret reagent: Sodium Potassium Tartrate, Sodium Hydroxide, Potassium Iodide ,Copper
Sulfate
 Standard: Protein solution (8 g/dl)
 Specimen/Sample: Serum, EDTA or Heparinized Plasma.
(Specimen Storage: Total protein is stable in serum and plasma for 1 week at room temperature, for
at least 1 month when refrigerated, at 2 to 8°C)

Method:

Sr. no. Reagent Test Standard Blank

1. Biuret 1 ml 1 ml 1 ml
2. Serum 0.02 ml ---- ----
3. Standard Solution ---- 0.02 ml ----
4. Distilled water ---- ---- 0.02 ml
Mix, incubate for 10 minutes at 20-25°C. Measure the absorbance of the Sample/Test and Standard
against the reagent blank at 546 nm , within 30 minutes.

Calculation:
Absorbance of test
Concentration of plasma protein (in g/dl) = --------------------------------- x Concentration of protein Standard
Absorbance of Standard
Normal Value:
Total plasma protein concentration (Adult) = 6.6 - 7.8 g/dl

[Plasma values are generally 0.3 to 0.5 g/dL higher than serum values due to the presence of fibrinogen.
This difference has been shown to vary among specific populations.]
Note: The values used in calculation are specific for the Human kit. These values may vary if other kits are used, however the
main principle will remain the same.

Clinical Significance:

 Hypoproteinemia
Hypoproteinemia may be caused by conditions including:
 nephrotic syndrome
 extensive bleeding
 sprue (deficient protein absorption)/malabsorption
 severe burns
 salt retention syndromes
 cirrhosis
 kidney damage
 Kwashiorkor (acute protein starvation)/malnutrition
 Hyperproteinemia
Hyperproteinemia may be caused by conditions including:
 severe dehydration
 multiple myeloma

Practical & Viva Questions

Q1. Which conditions cause Hypoproteinemia?
Hypoproteinemia may be caused by the following conditions:
 nephrotic syndrome
 extensive bleeding
 malabsorption
 severe burns
 salt retention syndromes
 cirrhosis
 kidney damage
 malnutrition
Q2. Which conditions cause Hyperproteinemia?
Hyperproteinemia may be caused by conditions including:
 severe dehydration
 multiple myeloma
  Severe exercise

Q3. What is the principle of this test?

The peptide bonds present in the protein react with copper sulfate in an alkaline medium to form a
purple/violet colored complex. Polypeptides containing at least two peptide bonds react with biuret
reagent. Cupric ion forms a coordination complex with protein nitrogen with very little difference
between albumin and globulin. The intensity of this color is proportional to the number of peptide
linkages present and thus is a measure of the concentration of proteins.