Rothera’s Test (Urine ketone bodies test) – Procedure, Principle and Results

Ketone bodies in urine is difficult to detect using basic urine testing procedure. Fortunately, there is a special test that has the ability to examine ketones in urine, and it is in the form of the Rothera Test.

It is a qualitative test used to detect ketone bodies in urine. Ketones are produced when fat is metabolized. The specific components include:

78% beta-hydroxybutyrate
20% acetoacetic acid
2% acetone. ^{(1, 2)}

For people with uncontrolled diabetes or those experiencing starvation, ketone synthesis takes place in the liver to be used as an energy source.

However, too high ketones in the bloodstream can lead to metabolic acidosis, which is a common scenario in people with diabetes.

Under normal circumstances, the extra level of ketones in the bloodstream is excreted from the body through urination, and too much ketone in urine is called ketonuria. ^{(2, 3)}

Principle of Rothera’s Test

Acetone and acetoacetic acid are reactive with alkaline solution present in sodium nitroprusside, which forms a purple-colored complex.

It has the ability to detect above 10-20 mg/dl of acetone and above 1-5 mg/dl of acetoacetic acid. However, it does not have the ability to detect beta-hydroxybutyrate. ^{(2, 3, and 4)}

Requirements and tools needed to perform the test

Urine sample
Test tube
Rothera powder consists of ammonium sulphate (20 grams) and sodium nitroprusside (0.75 grams)
Ammonium hydroxide/liquor ammonia ^{(3, 4, and 5)}

How to perform Rothera’s test?

Prepare a clean (sterile) test tube.
Pour a urine sample (5ml) into the test tube.
Add a mixture of Rothera’s powder (1 gram) to the test tube. Make sure the powder is thoroughly incorporated into the urine.
Once the mixture is thoroughly incorporated, the next step is to add 1 to 2 ml of concentrated ammonium hydroxide to the sample.
The added ammonium hydroxide creates a thin layer over the sample.
Watch out for a pink-purple ring that forms at the interface. ^{(5, 6, and 7)}

rothera rotheras test ketone bodies strip test

Interpreting Results

Positive Result

If you notice an instant appearance of a purple permanganate-colored ring, it indicates the presence of ketone bodies. Hence, the result is positive. ⁽³⁾

Negative Result

If you have not noticed the presence of a permanganate-colored ring at the interface, it means that the sample has no ketone bodies in it. Hence, the result is negative.

A test tube containing sample solution set for Rothera test. The presence of a purple permanganate-colored ring indicates a positive result

Picture 2 : A test tube containing sample solution set for Rothera test. The presence of a purple permanganate-colored ring indicates a positive result.

Safety precautions when performing Rothera Test

Make sure you thoroughly wash the apparatus, especially the one that needs to be sterile before use.
When handling chemicals, make sure you handle them with care to avoid any untoward incidents.
Make sure you do not get in contact with a urine sample while doing the experiment.
When handling the test tube, make sure you do not handle it with a bare hand, especially when there’s a specimen in it. Always use test tube holders.
Handle all the apparatus with care. put them in proper places after every use.^{(4, 7, 8, and 9)}

Frequently Asked Questions

Q1. What is the normal level of ketone?

The normal level of ketone in the blood is below 0.6mmol/L. If the level goes beyond 0.6, especially if it reaches 1.5mmol/L, it signals the possibility of DKA or diabetic ketoacidosis, a condition that if not managed right away can be fatal. ^{(6, 7)}

Q2. What ketone body is not detected by Rothera test?

Ketone bodies produced by the liver and used by the extrahepatic tissues are hardly detected by Rothera test because of their low concentration in urine. ⁽⁶⁾

Q3. What is the reason for the formation of purple ring in Rothera test?

The formation of purple ring (lavender-purple coloured complex) in Rothera test is caused by the reaction of acetoacetate or acetone with glycine to sodium nitroprusside. ⁽⁵⁾

Q4. What does the positive Rothera test indicate?

A positive Rothera test indicates the presence of ketones in urine, which is an affirmative sign in people with diabetes, especially if they are in a state of diabetic ketoacidosis, which is a fatal condition when not managed right away. ^{(4, 5)}

Q5. What tests are done to detect ketone bodies in urine?

There are various tests performed to detect the presence of ketones in the urine. Some of the most commonly used tests include Rothera’s test, which helps detect the presence of acetone and acetoacetic acid. Another reliable test is the ferric chloride test that helps check the level of acetoacetic acid, but it does not screen the presence of β-hydroxybutyric acid. Other tests worth mentioning include the reagent strip test and acetest tablet method. ^{(5, 6, and 7)}

Q6. Can you have ketones without having diabetes?

Yes, it is possible to have ketones in urine even if you do not have diabetes. It happens when the body uses fat as an energy source. Under normal circumstances, the body uses glucose as an energy source. However, in people experiencing starvation or those following a strict diet, the body relies on stored fat as an energy source. As the fat is broken down, one of its by-products is a ketone. Ketones in the urine can also be observed in people with an eating disorder, performing strenuous exercises, experiencing chronic vomiting, or following a low carbohydrate diet. ^{(4, 9, and 10)}

Q7. Why do diabetics have ketonuria?

The production of the ketone is high in people with diabetes and also in people who are fasting. It leads to increase production of ketone in the liver. The body gets rid of excess ketones in the body through urination, causing a high level of ketones in urine or also known as ketonuria. ^{(5, 6, and 8)}

Q8. Can Ketostix give a false positive?

Ketosix is one of the tests used to detect the level of ketones in the urine. However, the result is reliable only if the urine’s specific gravity is high, ranging between 1.010 and 1.020 and has a low pH level. However, there is also a possibility of a false-positive result when the urine being tested is highly pigmented. ^{(2, 5, and 9)}

References:

https://en.wikipedia.org/wiki/Ketone_bodies
https://www.ncbi.nlm.nih.gov/books/NBK247/
https://www.youtube.com/watch?v=7tcdQ5FkFX0
https://www.bioscience.com.pk/topics/pathology/clinical-pathology/item/822-tests-for-detection-of-ketones-in-urine
https://microbiologynote.com/rotheras-test/
http://laboratorytests.org/rotheras-test/
https://medicalstudyzone.com/rotheras-test-by-nitroprusside-reaction/
https://askinglot.com/what-is-the-principle-of-rotheras-test
https://academic.oup.com/ajcp/article-abstract/30/6/530/1762948?redirectedFrom=PDF
https://emedicine.medscape.com/article/2087982-overview