Heat and Acetic Acid Test (Urine test of proteinuria)

The majority of plasma proteins are large in particles making them hard to pass through the kidney’s glomeruli. However, smaller protein particles are filtered through and reabsorbed back into the blood by kidney tubules.

In normal urine conditions, traces of proteins are present such as Tamm-horsfall protein, hormones, mucopolysaccharides, albumin, enzymes, and immunoglobulins. An abnormally high level of protein in urine indicates renal disease as it signals abnormalities in tubular rate re-absorption.

The condition is called proteinuria, and one effective method of checking protein in urine is heat and acetic acid test. It is a qualitative method of checking proteins in urine. (1, 2, 3, and 4)


A comparison image of two test tubes with one tested negative for heat and acetic acid test tube tested positive image

Picture 1: A comparison image of two test tubes with one tested negative for heat and acetic acid test tube tested positive.

Principle of heat and acetic acid test

Heat and acetic acid test is based on proteins precipitating when exposed to an acidic medium. (4)

An actual image of a test tube containing urine tests for the presence of albumin pictures

Picture 2 : An actual image of a test tube containing urine tests for the presence of albumin.

How to perform heat and acetic acid tests?

  • Get a clean test tube and pour a clear urine sample (5 to 10 ml).
  • The upper part of the test tube filled with urine is brought to boil over a flame.
  • Once the upper part is heated, you have to compare it with the lower part. Look for signs of turbidity or cloudiness as it suggests the presence of carbonates, phosphates, or proteins.
  • A glacial acetic acid (10%), 2 to 4 drops are added to the test tube, and the upper part of the test tube is brought to boil for the second time.
  • Once again, check the upper part of the test tube and check for turbidity. If it disappears, it signals the presence of carbonates or phosphates. (4, 5, 6, and 7)

A qualitative analysis of urine using heat and acetic acid test images

Picture 3 : A qualitative analysis of urine using heat and acetic acid test.

Interpreting Results

The results are interpreted according to the grade of turbidity.

Negative – The result is negative when there’s no cloudiness observed.

Traces of protein – When there’s visible cloudiness, there are traces of protein present in urine. Traces of protein in urine are graded using below scaling system:

  • 1+ – Urine is cloudy but no granular
  • 2+ – There’s visible heavy cloudiness in the sample but no granular flocculation
  • 3+ – There’s visible dense cloudiness with visible flocculation
  • 4+ – Coagulation and thick curdy precipitation are visible. (7, 8, 9, and 10)


Frequently Asked Questions

Q1. Why is acetic acid used during heat coagulation tests?

In a urine sample with +3 protein, acetic acid, along with acetic buffer, test coagulates when exposed to high temperature (heat) and acid (protein denaturation). (3, 4)

Q2. Can albumin in urine be detected using a heat coagulation test?

Heat coagulation can detect albumin in urine by dropping a few drops of sulphosalicylic acid and bringing it to gentle heat. If you notice a cloudy or whitish turbid solution or precipitate(coagulation), it is a positive indicator of albumin in the urine. (8, 9)

Q3. What type of compound is detected in urine using the acetic acid test?

 Various compounds are detected in urine using an acetic acid test, such as ketone bodies. The formation of ß-hydroxybutyric causes the identity of these ketone bodies as a result of reduction and acetone secondary to decarboxylation. The aceto-acidic level ratio defines the number and type of ketone bodies in urine. (6, 7)

Q4. Can urine have acetic acid?

Acetic acid is present in urine. However, its number is high in people with urinary tract infections (UTI) for the reason that acetic acid is a bacterial metabolite. So, when there’s an increased amount of acetic acid in the urine, it signals urinary tract infection. (5)

  1. http://laboratorytests.org/heat-and-acetic-acid-test/
  2. https://www.bioscience.com.pk/topics/pathology/clinical-pathology/item/821-tests-for-detection-of-proteinuria
  3. https://www.youtube.com/watch?v=t2T8FqfPpE8
  4. https://microbenotes.com/heat-coagulation-test-of-proteins/
  5. https://www.scribd.com/doc/42099903/Acetic-Acid-Test
  6. https://nursingcrib.com/medical-laboratory-diagnostic-test/urinalysis-acetic-acid-method/
  7. https://pdfcoffee.com/acetic-acid-test-pdf-free.html
  8. https://www.histopathology.guru/a-chemical-examination-of-urine-protein-detection/
  9. https://www.ncbi.nlm.nih.gov/books/NBK705/
  10. http://edusanjalbiochemist.blogspot.com/2013/01/urinalysis-chemical-examination.html

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