Determination of Total Serum Proteins By Biuret Method

CASE HSTRORY A psychiatric patient was referred to the Psychiatric Clinic. His parents complained of his refusal to eat, loss of weight and edema of his feet.

Questions: Determine total serum proteins by Biuret method. What are the causes of hypoproteinaemia? What is the chemical basis for Biuret reaction? What is the use of blank and standard?

INTRODUCTION Plasma proteins include albumin, globulin and fibrinogen. They are mainly formed in liver. The normal level of plasma proteins range from 6 - 8 gm/dl.

Hypoproteinemia Levels less than 6 gm/dl indicate hypoproteinemia; e.g. in cases of: - loss of proteins through the skin (burns) - loss of proteins through urine (nephrotic syndrome) - Malabsorption - Decreased synthesis of plasma proteins (liver disease)

Hyperproteinemia Levels more than 8 gm/dl indicate hyperproteinemia; e.g. in case of: - multiple myeloma, - dehydration. - chronic diseases.

PRINCIPLE The purple color of the Biuret reaction is due to the complexing of Cu2+ in alkaline solution with the peptide bonds in the protein. The reaction takes place with compounds containing two or more peptide bonds, therefore, it can be used for quantitative determinations of peptides and proteins.

Standard solution (ml) Concentration 6.0 g/dl PROCEDURE 1. SET UP THREE TEST TUBES AS SHOWN IN TABLE:   Test Standard Blank Reagent\tube no. - 1.0 Standard solution (ml) Concentration 6.0 g/dl Unknown serum (ml) Dist. H2O (ml) 3.0 Biuret reagent (ml)

PROCEDURE (Cont.) 2. Mix and let the tubes stand at room temperature for 5 minutes. 3. Measure the absorbance at 520 nm, zeroing the instrument with blank. The color is stable for about 1-2 hrs after incubation. 4. Calculate the concentration of total proteins for the unknown. 5. Comment on your results.  

CALCULATION Reading of unknown Total Serum Protein Conc. = x Standard conc. (gm/dl) Reading of standard