Rothera Test for detecting Ketone bodies & its Procedure.

A urinalysis is an important tool that can help in the detection of various health conditions and diseases. Getting a urine analysis every month is a great way to stay up to date on your health status. Various findings can be deciphered from the urine tests of a person. One such test is the Rothera test. We will know more about the Rothera test throughout the article so keep going! 

What is ketonuria?

Ketone bodies are produced by the liver and are peripherally used when there is no other energy source available. As we are aware, the primary source of energy in humans is glucose. When there is a shortage of carbohydrates (glucose) in our body, the human body begins to extract energy from fats. This results in the production of ketone energy in the body. When the ketone bodies are generated in excess, they can be detected in the urine. This condition is known as Ketonuria.

Ketonuria often occurs in the following cases:

1. Diabetes Mellitus 
2. Weight loss
3. Starvation
4. Liver damage 
5. Anesthesia
6. Severe vomiting and diarrhea 
7. High fat intake or 
8. Reduced carbohydrate intake. 
9. Pregnancy.

Diabetes:

It often happens in Diabetes Mellitus patients that the body is unable to use stored glucose due to the low levels of insulin produced by the body. This pushes the body to obtain energy from stored fats and thus use ketone bodies as an energy source. Uncontrolled diabetes mellitus patients often suffer from ketonuria. The term used to describe this phenomenon is diabetic ketoacidosis. For those prone, it is advised to monitor their urine for the buildup of ketone bodies.

Weight loss:

People who are trying to lose weight often go on diets that forbid them from carb-loading or expect them to cut carbohydrates completely. Some people prefer intermittent fasting or fad diets like eating one meal a day. These diet changes can put their body into the process of limited starvation and thus into ketosis. The presence of ketones in the urine of people undergoing a weight loss journey is common. 

Liver damage:

Liver damage due to diseases like NAFLD can lead to ketone bodies in the urine. Severe occurrences of diarrhea, vomiting, or indigestion can also cause disturbances in the body’s metabolism and lead to ketonuria. 

Anesthesia:

People undergoing surgical procedures like bypass operations or any other surgical procedure need anesthesia. It has been observed that post-operative patients are often tested positive for ketonuria. This can be because of a number of factors. As per research, it could be because of the use of anesthetic gas that can lead to hepatic changes. An increase in liver fat or decrease in liver glycogen during or post-operation can also be the main reason for ketones in the urine. Having an adrenaline rush during the procedure can also lead to the flow of ketone bodies into the urine.  Deprivation of food for operative patients can sometimes lead to a state of starvation and thus result in ketonuria.

Extreme diets:

Following a turbulent diet with a high fat intake can lead to the metabolism of fats and the flow of excessive ketones into the urine. Whereas a low-carb diet can force the body to metabolize fats and produce ketones, the traces of which might appear in the urine. People with eating disorders like anorexia and bulimia can fall prey to further health complications due to ketonuria.  

Pregnancy:

A normal pregnant woman may or may not face ketonuria. However, Certain physiological and hormonal changes can trigger ketonuria in pregnant women. Certain pregnant ladies that suffer from abnormal insulin secretion and metabolism issues are at a greater risk of ketonuria. 

What is the Rothera test?

Rothera test is a lab test that helps in the detection of ketones in human urine samples. Talking about ketones, there are 3 types of ketones/ketone bodies/ acetone bodies that can be found in the urine. On the metabolism of fats, 3 ketonic products can be found in the urine namely acetone (2%), acetoacetic acid (20%), and beta-hydroxybutyrate (78%).

No method of testing for the detection of ketone bodies reacts with all 3 of these compounds. The Rothera test works by detecting the presence of acetoacetic acid and acetone in the urine. No screening test can confirm the presence of β-hydroxybutyric acid in the urine. The Rothera test is 10-20 times more sensitive to acetoacetic acid than acetone. 

Principle of the Rothera test:

Rothera test is a method for testing the presence of ketones in the urine. A solution of strong ammonia is added to a sample of urine followed by sodium nitroprusside, This leads to the formation of a purple-colored complex detecting the presence of acetoacetic acid and acetone. Rothera’s test is sensitive to 1-5 mg/dl of acetoacetate and 10-25 mg/dl of acetone. The presence of β-hydroxybutyric acid is not detected in the urine.

Aim of the Rothera test:

To detect the presence of ketone bodies in the urine and confirm ketonuria.

Requirements for the screening test:

Following requirements are essential for performing the test.

1. Urine specimen
2. Glassware: test tube, pipette
3. Chemicals: 

1. Concentrated Ammonium hydroxide (liquor ammonia)
2. Rothera’s powder: Sodium nitroprusside = 0.75 gm, Ammonium sulphate = 20gm (Mix and pulverize).

The procedure for the Rothera test:

To perform this screening test, 

1. Wear gloves and a mask for personal safety.
2. Take around 5ml of the urine sample in a clean test tube.
3. To this add 1g of Rothera’s powder and mix well by shaking the test tube.
4. To this mixture gently add 1-2ml of concentrated ammonium hydroxide.
5. Look for the formation of a purple-pink ring at the interface of the two liquids. 

Observation if the Rothera test is positive:

If the specimen urine sample contains the ketone bodies- acetoacetic acid and acetone then there will be the presence of a purple-pink-colored ring in the test tube. This means the Rothera test is positive for this urine sample. 

Observation if the Rothera test is negative:

If the specimen urine sample does not contain ketone bodies, there will be no change of color in the test tube. This means the Rothera test is negative for the sample.

Precautions while performing the Rothera test:

Keep in mind these precautions for personal safety while performing the test

1. Wear gloves and masks while performing the test.
2. Do not touch the test tube with your hand, use a test tube holder for this purpose.
3. Be careful with the chemicals in the laboratory.
4. Use clean test tubes to perform the tests as not doing so might affect the results of the test.
5. Use chemicals in the same quantities as mentioned in the procedure.
6. Wash the apparatus before and after performing the tests.

What are the different methods of detecting ketonuria? 

Other methods for detecting ketone bodies in the urine are listed below.

1. Gerhardt’s test 
2. Lang’s test 
3. Lindeman’s test
4. Han’s test and 
5. Acetest tablet test
6. Reagent strip test

All the above-mentioned tests might have slight deviations but they all follow the same principle of the Rothera test. 

Who Should Go for the Rothera test?

RUA or routine urine analysis is recommended for anyone who is getting a daily health checkup. A healthcare provider might insist on a urine test like the Rothera test if they suspect a health condition like diabetes. People with pre-existing health conditions like Diabetes Mellitus, high blood pressure, eating disorders like anorexia or bulimia, digestive issues, vomiting, and diarrhea,  liver disease, being Pregnant, etc are some of the conditions that may require the person to get a urine examination along with a Rothera test.

How accurate is the Rothera test?

The Rothera test is a highly accurate test when the urine sample is tested with a high specific gravity (between 1.010 and 1.020) and low-pH. A highly pigmented urine specimen that is dark yellow or brown in color may yield a false positive reading. Certain drugs can also interfere with the accuracy of the test. It is a possibility that Levodopa can yield a false-positive result for the test. With several new methods available for measuring ketone bodies, with the isolation and purification of the enzyme 3-hydroxybutyrate dehydrogenase, it is now possible to develop a sensitive and highly specific enzymatic assay for acetoacetate detection. Newly developed chromatographic techniques for the detection of both acetoacetate and acetone in the urine are now available. Its application for the detection of hydroxybutyrate is also possible according to its researchers. 

What are the advantages of the Rothera test?

Following are the reasons why the Rothera test is one of the most preferred techniques for the detection of ketone bodies in the urine.

1. Accurate.
2. Limited chemicals are required.
3. Cost-effective.
4. Quick.

So update your knowledge about the latest and the most accurate tests in diagnostic today, Happy learning!

References:

1. Laffel L, et al. Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes. Diabetes Metab Res Rev. 1999 Nov-Dec;15(6):412-26.
2. Hunter A, et al. “Ketosis of anaesthesia.” Canadian Anaesthetists’ Society Journal 2.4 (1955): 308-318.
3. Qian, Meichen et al. “Effect of Elevated Ketone Body on Maternal and Infant Outcome of Pregnant Women with Abnormal Glucose Metabolism During Pregnancy.” Diabetes, metabolic syndrome and obesity : targets and therapy vol. 13 4581-4588. 25 Nov. 2020.
4. Rothera’s test Principle, Procedure, Result. at https://microbiologynote.com/rotheras-test/  
5. Comstock JP, et al. Ketonuria. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990. Chapter 140.

FAQs