Blood Protein Determination

Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
 Objectives
• At the end of this session, the student should be able to:
1. Identify the principle involved in the determination of total serum
protein and albumin/globulin ratio;
2. Correlate the laboratory findings with clinic-pathological state;
3. Observe the practical consideration in performing the test.

Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
 Introduction
• Proteins are the chief nitrogenous components of cellular structures.
• They have been described as the “essence of life processes”.
• Proteins are polymers of alpha amino acids linked together by peptide bonds.
• The enzymatic reaction sequence employed in the assay of total protein as follows:

Protein + Cu++ Alkaline Cu-protein complex 

pH
• The intensity of the color is proportional to the amount of protein present when compared
to a solution with known protein concentration.

Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
I. Total Serum Proteins
  Methods of Analysis
1.1 Kjeldahl Method
Principle: Digestion with sulfuric acid in the presence of catalysts converts an forms of nitrogen in the
specimen to ammonium ions which is then separated from the digestant by steam distillation into boric
acid solution. The ammonium ions are then quantitated by any of the following:

•Titration with a standard solution of HCI

•Photometrically with Nessler's reagent 
•Berthelot reaction

1.2 Biuret Method 

•Principle: Formulation of violet-colored complex between cupric ions and peptide bonds in alkaline
medium. The intensity of violet color is related positively to the concentration of protein.

Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
I. Total Serum Proteins
  Methods of Analysis
1.3 Lowry Oxidation Method 
•Principle: Pre-treatment with alkaline copper solution followed by addition of folin and
ceocaltein/phenol reagent which oxidized the phenolic structure of amino acids. The deep color
produced results from the reduction of phosphotungstic and phosphomolybdic acids to molybdenum
blue and tungsten blue.
 
1.4 Refractometry 
•Measurement of refractive index of dissolved solids.

1.5 Ultraviolet Absorption

•Principle: Light absorption of peptide bonds due to tryptophan and to a lesser degree into
phenylalanine and tyrosine at 210 nm.
Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
I. Total Serum Proteins
  Methods of Analysis
1.6 Turbidimetric Method
•Principle: Proteins are precipitated using sulfosalicylic acid, trichloroacetic acid or acetic acid
-potassium ferrocyanide solution and quantitated using Biuret reaction.

1.7 Precipitation
•Principle: Changes of net charge of protein resulted from precipitation

Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
•Total serum protein and albumin/globulin ratio are determined when peptide bonds of
proteins react with cuprous ions in alkaline medium to form a colored product where the
absorbance is measured spectrophotometrically at 540 nm.
•In this method, a measured amount of serum is treated with 23% sodium sulfate. An aliquot
portion of the mixture is set aside for the total protein determination. The remaining mixture is
then treated with ether. Globulin is precipitated at the junction of che two layers. An aliquot
portion of the aqueous (lower layer) is set aside for albumin determination. The portion is
then treated with special Biuret reagent. From the total serum protein and albumin values,
the globulin can be estimated using the difference. The method described permits the
estimation of total proteins, albumin and globulin on a single sample.
•The Biuret reagent contains sodium potassium tartrate salt to complex cupric ions and
maintains solubility in alkaline solution. Biuret reagent is dimmer produced by heating urea.
The Biuret reaction is given by compounds with at least two amino bonds or peptide bonds.
In a strongly alkaline medium, Biuret reagent gives a violet color with copper sulfate in the
presence of proteins.

Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
I. Total Serum Proteins
  Methods of Analysis
1.6 Electrophoresis
Principle: Migration of charged particles in an electric field.
Clinical application: Identification of monoclonal spike of immunoglobulin

Reference values for each Fraction:

1. Albumin – 53% to 65%
2. a1 globulin – 2.5 – 5%
3. a2 globulin – 7-13%
4. B-globulin – 8 – 14%
5. y-globulin – 12 – 22%

Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual
Source: Suba, S.,& Florida, J., (2014)., Blood Chemistry 1 Laboratory Manual