Estimation of Plasma Proteins by the Biuret Method

Here is a full explanation of everything shown in your notes:

1. Principle

Proteins have peptide bonds (-CO-NH-) in their backbone. When two or more peptide bonds are present, the protein reacts with cupric ions (Cu²+) in an alkaline solution to form a blue-to-purple colored complex.

The method is named after Biuret (NH₂CONH CONH₂), a simple molecule with two CO-NH groups that gives the same color reaction.
The intensity of the purple color is directly proportional to the protein concentration - this is the basis for quantification using a colorimeter/spectrophotometer.

In short: More protein = more peptide bonds = more color = higher absorbance reading.

2. Reagents

Reagent	Role
Copper sulphate (CuSO₄)	Provides Cu²+ ions that form the colored complex
Sodium hydroxide (NaOH)	Creates the alkaline environment needed for the reaction
Sodium potassium tartrate	Stabilizes Cu²+ ions in solution (prevents precipitation)
Potassium iodide (KI)	Prevents auto-reduction of copper; acts as a preservative
Standard Human Albumin (8 gm%)	Known concentration used for comparison/calibration

3. Procedure - The Three-Tube Setup

Reagent	Blank	Standard	Test
Biuret reagent	✓	✓	✓
Working standard (albumin)	-	✓	-
Test sample (patient serum)	-	-	✓
Distilled water	✓	-	-

Blank = zero protein reference; used to zero the machine
Standard = known protein concentration (8 gm%); used to calibrate
Test = unknown patient sample whose protein you are measuring

Incubate all three at 37°C for 10 minutes, then read optical densities (OD).

4. Calculation

$$\text{Protein (Test)} = \frac{\text{OD}_T - \text{OD}_B}{\text{OD}_S - \text{OD}_B} \times \text{Concentration of Standard}$$

Where:

OD_B = Optical Density of Blank
OD_S = Optical Density of Standard
OD_T = Optical Density of Test (patient sample)

Why subtract OD_B? The blank corrects for background color from the reagent itself (non-protein color). You always subtract blank from both test and standard to get true protein-related absorbance.

5. Normal Reference Ranges

Protein Fraction	Normal Range
Total Protein	6.3 - 8.4 gm/dL
Albumin	3.5 - 5.8 gm/dL
Globulin	2.5 - 3.5 gm/dL

Important note: Albumin specifically is estimated by the BCG (Bromocresol Green) method, not Biuret. Biuret measures total protein.

6. Clinical Interpretation

Hyperproteinemia (Raised protein - above normal range)

Category	Cause	Mechanism
Hemoconcentration	Vomiting, diarrhea	Loss of water/fluids → proteins become relatively concentrated
Physiological	Standing, exercise, tourniquet	Fluid shifts out of vasculature → proteins concentrate temporarily
Pathological	Multiple myeloma	Abnormal immunoglobulin production by malignant plasma cells
Pathological	Tuberculosis	Chronic infection → raised globulins (immune response)

Hypoproteinemia (Decreased protein)

The bottom of the image is partially cut off, but common causes include: malnutrition, liver disease (reduced albumin synthesis), nephrotic syndrome (protein loss in urine), malabsorption, and burns.

Key Points to Remember

Biuret = total protein measurement; requires at least 2 peptide bonds.
Purple color = Cu²+ forms a coordination complex with 4 peptide nitrogens.
Albumin separately estimated by BCG (Bromocresol) method.
The formula uses the standard as a calibrator - no need for a standard curve.
Multiple myeloma is the classic pathological cause of raised total protein (due to excess paraprotein/M-protein).

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