Heat Coagulation Test of Proteins – Principles, Procedure, Result

Heat coagulation test of proteins is a qualitative biochemical test used to detect the presence of proteins mainly albumin and globulin in urine. It is based on the property of proteins to undergo denaturation on heating. When urine containing protein is heated the internal bonds of protein is broken and the polypeptide chains are unfolded and aggregated to form an insoluble mass which is referred to as coagulum. This coagulation is maximum at isoelectric point of protein therefore the urine is usually acidified with weak acid like acetic acid before or during the test.

In this test the urine sample is taken in a test tube and only the upper portion is heated to boiling while the lower portion is kept unheated which acts as control. If turbidity or cloudiness appears in the heated part it indicates either protein or phosphates. To differentiate this a few drops of acetic acid is added. If the turbidity persists or increases it confirms the presence of protein whereas turbidity due to phosphates disappears. Most proteins form permanent coagulum on boiling but Bence-Jones protein shows a characteristic behavior where it precipitates at 40–60°C dissolves on boiling and reappears on cooling.

Objectives of Heat Coagulation Test of Proteins

• To detect the presence of proteins in urine mainly albumin and globulin.
• To demonstrate denaturation of proteins by heat resulting in formation of coagulum.
• To differentiate protein turbidity from inorganic salts like phosphates and carbonates by using acetic acid.
• To detect Bence–Jones protein which shows characteristic precipitation and dissolution on heating and cooling.
• To screen proteinuria in clinical conditions such as kidney disorders and pre-eclampsia.
• To demonstrate that coagulation of protein occurs maximum at isoelectric point of protein.
• To distinguish coagulable proteins from non-coagulable substances like peptones and proteoses.

Principle of Heat coagulation test of proteins

The principle of heat coagulation test of proteins is based on the denaturation of protein by heat. When urine containing proteins is heated the secondary and tertiary structure of protein is destroyed due to breaking of hydrogen bonds and other weak bonds. As a result the polypeptide chains are unfolded and aggregated together to form an insoluble semi solid mass which is known as coagulum. This process is irreversible in nature and indicates the presence of protein in the given sample.

This coagulation of protein occurs maximum at the isoelectric point where the protein carries no net electrical charge and the repulsive forces between molecules is minimum. For this reason dilute acetic acid is added to adjust the pH of urine around 5.4 which is the isoelectric point of albumin. The acid also helps in dissolving inorganic precipitates like phosphates and carbonates which may produce turbidity on heating. Therefore persistence of turbidity after addition of acetic acid confirms that the coagulation is due to protein and not due to inorganic salts.

Requirements for Heat Coagulation Test of Proteins

• Test tube – clean and dry glass test tube.
• Test tube holder – to hold the test tube during heating.
• Heat source – Bunsen burner or spirit lamp.
• Dropper or pipette – for adding reagents.
• Urine sample – fresh urine sample preferably clear and filtered.
• Acetic acid – 1% to 3% acetic acid solution.
• Chlorophenol red indicator (optional) – to adjust pH near isoelectric point.
• White paper or black background – for observing turbidity clearly.

Procedure of Heat Coagulation Test of Proteins

• Take a clean and dry test tube and fill it about two-thirds with urine sample.
• Hold the test tube in an inclined position using a test tube holder.
• Heat only the upper portion of the urine gently over a flame until it starts boiling.
• Observe the heated upper portion and compare it with the unheated lower portion which acts as control.
• Note the appearance of turbidity or coagulum in the heated part.
• Add 3–5 drops of dilute acetic acid to the heated urine.
• Observe the urine again for persistence or disappearance of turbidity.
• Persistence or increase in turbidity indicates presence of protein while disappearance indicates inorganic salts.

Result and Interpretation of Heat coagulation test of proteins

General Observations

• Negative result
  If the upper heated portion of urine remains clear it indicates absence of coagulable proteins.
• Positive result
  If cloudiness or white coagulum is formed on heating and it persists or increases after addition of acetic acid it indicates presence of proteins like albumin and globulin.
• False positive (Phosphates / Carbonates)
  If cloudiness appears on heating but disappears completely after adding acetic acid it is due to inorganic salts like phosphates or carbonates and not due to proteins.
  If cloudiness disappears with effervescence (gas formation) it indicates presence of calcium carbonates.

Grading of Proteinuria (Semi-quantitative Interpretation)
If the test is positive the amount of protein is roughly interpreted based on intensity of turbidity.

• Trace – Mere haziness or slight opalescence is seen (around 10 mg/dL).
• 1+ – Distinct cloudiness is formed but printed text can be read through the test tube (about 10–50 mg/dL).
• 2+ – Heavy or granular cloudiness is seen and printed text appears as faint lines only (about 50–200 mg/dL).
• 3+ – Dense turbidity with flocculation is formed and printed text is not visible (about 200–500 mg/dL).
• 4+ – Thick heavy coagulum or massive precipitate is formed (more than 500 mg/dL).

Special Interpretation (Bence-Jones Proteins)

• If white precipitate is formed between 40°C–60°C disappears on boiling at 100°C and reappears on cooling it indicates presence of Bence-Jones proteins.
• This type of reaction is associated with multiple myeloma and related plasma cell disorders.

Uses of Heat Coagulation Test of Proteins

• It is used for detection of proteins mainly albumin and globulin in urine and is helpful in screening of proteinuria.
• It is used as a routine qualitative test for assessing renal disorders where leakage of proteins occurs in urine.
• It is used in diagnosis of pre-eclampsia in pregnancy by detecting proteinuria in urine samples.
• It is used for detection of Bence-Jones proteins which are associated with multiple myeloma and related plasma cell disorders.
• It is used to differentiate protein turbidity from turbidity caused by inorganic salts like phosphates and carbonates with the help of acetic acid.
• It helps in distinguishing heat coagulable proteins such as albumin and globulin from non-coagulable substances like peptones and proteoses.
• It is used in teaching laboratories to demonstrate protein denaturation and heat coagulation at isoelectric point.
• It is used as a simple and cost-effective screening test in laboratories with limited facilities.

Limitations of Heat Coagulation Test of Proteins

• It is a subjective test as the result depends on visual observation of turbidity and may vary from person to person.
• It may give false negative result in alkaline urine if proper acidification is not done before heating.
• In alkaline condition proteins may form alkaline metaprotein which does not coagulate on heating.
• It may give false positive result due to precipitation of phosphates and carbonates on heating.
• Mucin present in urine may produce turbidity and cause error in interpretation.
• It is not a truly quantitative test and gives only qualitative or semi-quantitative estimation of protein.
• The result may vary with concentration of urine as dilute urine may mask protein while concentrated urine may exaggerate it.
• It is less sensitive compared to sulfosalicylic acid test and modern dipstick methods.
• It does not detect non-coagulable proteins like peptones proteoses and gelatin.
• Bence-Jones proteins may be missed if precipitation at lower temperature is not observed carefully.
• High amount of albumin may mask the reaction of Bence-Jones proteins.
• It is less convenient and relatively hazardous as it requires heating and use of acids.

Advantages of Heat Coagulation Test of Proteins

• It is a simple and economical test and does not require costly reagents or instruments.
• The procedure is easy to perform and does not need special technical skill.
• It gives fairly specific results with comparatively less false positive reactions.
• It helps in differentiating protein turbidity from turbidity caused by phosphates and carbonates by addition of acid.
• It is suitable for routine screening of urine samples in basic laboratories.
• It is useful in laboratories with limited facilities and in peripheral health centers.
• It is reliable for detection of moderate to severe proteinuria.
• It can be performed with minimal apparatus like test tube heat source and acetic acid.