Rothera’s Test: Procedure, Principle, Results and Clinical significance

Rothera’s test is used to detect ketone bodies in urine, which are by-products of fat metabolism in the body. These by-products include acetoacetic acid (2%), beta-hydroxybutyric acid (78%), and acetone (2%). Acetoacetic acid and acetone are commonly referred to as true ketone bodies.

This test is qualitative, meaning it shows whether ketones are present or not. It is useful in diagnosing conditions like ketonemia (ketones in the blood), ketonuria (ketones in urine), hyperemesis gravidarum (severe nausea and vomiting in pregnancy), and ketoacidosis, a serious condition often seen in diabetes mellitus, fasting, or starvation when the body breaks down fats due to low glucose levels.

Ketone bodies are not attached to plasma proteins, so they can be freely filtered by the kidneys. Some are reabsorbed back into the bloodstream, while any excess is excreted in the urine.

Rothera’s test principle

Rothera’s test detects the presence of ketones, specifically acetoacetic acid and acetone, in urine. These substances react with sodium nitroprusside in an alkaline environment, such as ammonium hydroxide, to produce a purple color. This color change indicates the presence of ketones.

However, beta-hydroxybutyrate, another ketone body, does not react in this test because it lacks a specific chemical group called a keto group. Despite this, its presence can still be inferred because beta-hydroxybutyrate is in equilibrium with acetoacetic acid, meaning that some of it may convert into acetoacetic acid, which can then be detected by the test.

Rothera’s test is sensitive enough to detect as little as 5 mg/dl of acetoacetic acid and 10 mg/dl of acetone in urine, making it a useful tool for identifying even small amounts of ketones.

The requirements of the Rothera’s tests

To perform Rothera’s test the following are required:

• Urine specimen to be tested
• Test tubes to hold the urine sample during testing
• Rothera’s powder which is a mixture of 0.75g sodium nitroprusside and 20g ammonium sulfate.
• Ammonium hydroxide also known as liquid ammonia provides an alkaline environment for the test.

Rothera’s test procedure

1. Ensure all equipment is clean and sterile.
2. Pour 5 ml of the urine sample into a clean test tube.
3. Add 1 gram of Rothera powder to the urine and mix thoroughly.
4. Carefully pour 1-2 ml of ammonium hydroxide on top of the urine mixture without stirring.
5. Watch for a color change at the point where the urine mixture and ammonium hydroxide meet.

Interpretation of Rothera test results

Rothera’s test is semi-quantitative in that it does not provide the exact level of ketones but the depth of color change can provide a rough estimate of the concentration.

The immediate formation of a purple permanganate ring at the interface of the urine-ammonium hydroxide mixture signifies a positive result. Ketone bodies are present.

A negative result is indicated by no colored ring formation. Ketone bodies are absent.

Since color intensity may vary at the interface, positive results can be quantified and graded as

• Low concentration                  +
• Moderate concentration         ++
• High concentration                  +++
• Very high concentration          ++++

Other tests for ketone bodies include:

1. Blood tests: Similar to testing blood glucose with a finger prick. These tests are more accurate than urine ketone tests.
2. Breath ketone test: Detects acetone in the breath.
3. Dipstick test: A strip is dipped into a urine sample, and the color change is compared to a chart to determine the presence of ketones.
4. Gerhardt’s test: Involves a reaction between ferric chloride and acetoacetic acid, resulting in a Bordeaux red color change.
5. Lang’s Test: This test involves adding a few drops of urine to a solution of sodium nitroprusside. If ketones are present, the solution will change color, typically to a deep purple or red.
6. Lindeman’s Test: In this test, a urine sample is treated with a solution of iodine and potassium iodide. The presence of ketones results in a yellow or brown precipitate forming in the solution.
7. Han’s Test: This test involves mixing a urine sample with ammonium sulfate and sodium nitroprusside. If ketones are present, the solution will turn red or purple.
8. Tablet Test: A commercially available tablet containing sodium nitroprusside and glycine is added to a urine sample. If ketones are present, the tablet will cause the solution to change color, usually to purple or red, indicating a positive result.

Rothera’s test is crucial in detecting ketone bodies in urine and therefore a vital test in diagnosing different conditions in diabetes management, pregnancy, and during starvation and fasting. However, it should be done with other tests and clinical judgment to get the exact concentration of ketones and accurate diagnosis and treatment. Other tests can also be used in the detection of ketones