Introduction
Total Proteins are the most abundant compounds in serum. Amino acids are the building blocks of all proteins. In turn, proteins are the building blocks of all cells and body tissues. They are the basic components of enzymes, many hormones, antibodies and clotting agents. Proteins act as transport substances for hormones, vitamins, minerals, lipids and other compounds.
Plasma/serum proteins comprise a complex mixture of different proteins. The important total proteins in the plasma/serum are:
- Albumin
- Globulin
- Conjugated proteins such as lipoproteins
- Fibrinogen (absent from serum)
Methods for Estimating Proteins
- Nitrogen estimation by Kjeldahl technique
- Ninhydrin reaction
- Lowry’s method for estimating tyrosine estimation
- Biuret method
Method
The Biuret method.
Principle
Substances that contain two CONH2 groups joined together directly or through a single carbon or nitrogen atom, and those that contain two or more peptide links give a purple-coloured compound with an alkaline copper solution.
Reagents
- Stock biuret reagent
- Biuret solution for use
- Tartrate iodide solution
- Bovine or human albumin standard, 2 mg/ml.
Procedure
| Test | Standard | Blank | |
| Serum, ml | 0.1 | – | – |
| Albumin standard, ml | – | 3.0 | – |
| Distilled water, ml | 2.9 | – | 3.0 |
| Biuret reagent, ml | 3.0 | 3.0 | 3.0 |
Mix and incubate all the tubes at room temperature for 10 minutes. Read absorbance against reagent blank at 540 nm or green filter.
Calculation
Concentration of Total Protein in 100 ml serum (g %)
= T/S × con of std × 100/vol. of serum × 1/1000
= T/S × 5 mg × 100/0.1 × 1/1000
= T/S × 5 g%
Concentration of standard = 5 g%
= 5000 mg/ 100 ml
= 500 mg/10 ml
= 50 mg/1 ml
= 5mg/ 0.1 ml
Normal Values
- Total serum proteins – 6–8 g/dl
- Albumin – 3.7–5.3 g/dl
- Globulin – 1.8–3.6 g/dl
Clinical Significance
Total protein increased due to:
- Chronic infection (including tuberculosis)
- Adrenal cortical hypofunction
- Collagen vascular disease
- Hypersensitivity states
- Sarcoidosis
- Dehydration
- Respiratory distress
- Haemolysis
- Cryoglobulinemia
- Alcoholism
- Leukaemia
Total protein decreased due to:
- Malnutrition and malabsorption
- Liver disease
- Diarrhoea
- Severe burns
- Hormone imbalances that favour the breakdown of tissue
- Low albumin
- Low globulins
- Pregnancy