Biochemical Test

Rothera’s test Principle, Procedure, Result.

Acetoacetic acid and acetone react with an alkaline solution of sodium nitroprusside to form a purple-colored complex. This method can detect above...

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This article writter by MN Editors on July 19, 2021

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Rothera’s Test
Rothera’s Test

Rothera’s test Definition

  • Rothera’s test is a type of laboratory test which is performed for the qualitative detection of ketone bodies in urine.
  • During the “ketosis” three ketones bodies or acetone bodies are found in the urine which are the products of fat metabolism, known as acetone (2%), acetoacetic acid (20%) and beta-hydroxybutyrate (78%).
  • During starvation and uncontrolled diabetes, the Ketone bodies are synthesized within the liver to re-utilized energy. These are types of acid that can results in metabolic acidosis in uncontrolled diabetes.
  • If the rate of ketone bodies production exceeds, the excess amounts of ketone bodies will be eliminated through the urine and the condition is termed as ketonuria. Acetone is volatile and also excreted in breath.
  • Ketosis may be correlated with diabetes mellitus termed Diabetic ketoacidosis, or it may be due to starvation, persistent vomiting and high fat and low carbohydrate diet.
  • There are present different methods for the detection of ketones in urine such as Rothera’s test, Gerhardt’s test, Lang’s test, Lindeman’s test, Han’s test, and Tablet test. All the tests used for the detection of ketonuria are based on the principle of Rothera’s nitroprusside test.

Aim of Rothera’s Test

To detect the presence of Ketone bodies within the supplied urine sample.

Rothera’s Test Principle

Acetoacetic acid and acetone react with an alkaline solution of sodium nitroprusside to form a purple-colored complex. This method can detect above 1-5 mg/dl of acetoacetic acid and 10-20 mg/dl of acetone. Beta-hydroxybutyrate is not detected.

Requirement for Rothera’s Test

  • Specimen: Urine 
  • Glassware: Test tubes, pipette.
  • Rothera’s powder: Sodium nitroprusside = 0.75 gm, Ammonium sulphate = 20gm (Mix and pulverize).
  • Liquor Ammonia (Ammonium hydroxide)

Rothera’s Test Procedure

  1. Take a clean test tube and add 5 ml of urine to it.
  2. Transfers  1 gm of Rothera’s powder mixture within the test tube and mix well.
  3. Add 1-2 ml of concentrated ammonium hydroxide to the urine sample within the test tube. It will create a thin layer over the urine sample.
  4. Observe the pink-purple ring at the interface.

Results

  • Positive Test: If a purple permanganate-colored ring is immediately formed at the interface, means Ketone bodies are present.
  • Negative Test: If no permanganate colored ring is formed at the interface, means ketone bodies are absent within the test sample.
Rothera’s Test
Rothera’s Test

Precautions

  • Wash the apparatus before and then after the experiment.
  • Carefully manage all the chemicals within the laboratory.
  • Don’t touch the urine sample during the experiment.
  • Holds the test tubes by using test tube holders.
  • Use a clear and clean test tube, make sure the test tubes are free of any dirt and chemicals because this will give a false result.
  • Place all the apparatus in their respective place after the completion of the experiment.
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