Under normal conditions, metabolized fats are completely broken down to water and CO2, hence negligible amount (1mg/24hr) of ketone bodies are excreted in urine.
When the rate of production is increased, excess of ketone bodies are eliminated in urine
3. What are the tests for ketone bodies in urine
Rothera’s test
Ketostrip test
Gerhardts test (Diacetic acid, acetoacetic acid)
Lindeman test (Diacetic test)
Harts test ( β- hydroxy butyric acid)
4. What is the principle of Rotheras test
Ketone bodies (acetone or acetoacetic acid) develop a purple colored complex with sodium nitroprusside in an alkaline solution
This method can detect only acetone and acetoacetic acid but β- hydroxy butyric acid cannot be detected
5. What is the procedure of Rotheras test
Take 5 ml of urine in test tube
Saturate it with crystals of ammonium sulfate
Add 2 drops of freshly prepared sodium nitroprusside solution or crystal of sodium nitroprusside and mix well
Add 2 to 3 ml of concentrated Ammonia along the sides of the test tube so that it forms a layer on the top of saturated urine
Positive test shows reddish purple ring at the interface
6. How to detect β- hydroxy butyric acid
It cannot be detected by routine test
It should be oxidized by adding H2O2 to acetoacetic acid and then tested
Procedure
Add 2 – 3 drops of acetic acid to 5 – 6ml of 1:10 diluted urine
Boil it for 3 – 4 minutes so that acetone and acetoacetic acid are removed
Then add 1ml of H2O2 and warm gently
Cool it and later perform Rotheras test
7. What is the principle of Gerhardt’s test
Ferric chloride reacts with acetoacetic acid to form a portwine or Bordeaux red color. This test detects only acetoacetic acid. Acetone and β- hydroxy butyric acid cannot be detected
8. Explain the procedure of Gerhardt’s test
Take 5 ml of urine in test tube
Add 5ml of 10% Ferric chloride drop by drop to the urine
If phosphates are present, they get precipitated as Ferric phosphates
On further addition of excess of Ferric chloride, Bordeaux red color develops if acetoacetic acid is present
To confirm the presence of acetoacetic acid, boil the test solution for 5 minutes
If color disappears on boiling, it indicates the presence of acetoacetic acid
If color persists, it indicates absence absence of acetoacetic acid and presence of salicylates
Color disappears on boiling as acetoacetic acid loses CO2 and is converted to acetone which cannot be detected as it does not react with Ferric chloride
9. What are the conditions with Ketonuria
Diabetes mellitus
Starvation
Prolonged vomitting
High fat and low carbohydrate diet
prolonged febrile illness
Toxemia of pregnancy
Glycogen storage disease
10. What is the principle of Hart’s method ?
This is a method for converting Beta hydroxy Butyric acid to Diacetic acid using Hydrogen peroxide, after first eliminating acetone and Diacetic acid byboiling and then testing with reagent nitroprusside
By
Dr.Shanthi Vissa, Associate professor, Pathology, Sri Venkateswara Institute Of Medical Sciences, Tirupathi.
Dr. Byna Shyam Sundar Rao, Professor, Pathology, Narayana Medical College, Nellore