Test for Ketone bodies in Urine (Gerhardt’s test)

Test for ketone bodies in urine (Gerhardt’s test) Principle

The Gerhardt’s test is a test that measures the qualitative amount of ketone bodies in the urine. Ketone bodies are of three types namely acetone, acetoacetic acid and Beta-Hydroxybutyric acid. When ketosis occurs due to fat metabolism, urine contains 2% acetone, 20% acetoacetic acid, and 78% beta-hydroxybutyric acid. Only the presence of acetoacetic acid can be known with the help of Gerhardt’s test.

The amount of fat metabolism that occurs under normal conditions is completely broken down to produce water and carbon dioxide. As a result, very small amounts of ketone bodies are excreted in the urine. But when fat metabolism becomes excessive, the production of ketone bodies increases. This causes a large amount of ketone bodies to be excreted in the urine.

To perform this test, a few drops of 10% ferric chloride (FeCl₃) is added with urine sample. The formation of cherry or reddish red color indicates the presence of acetoacetic acid (CH₃COCH₂COOH) in the urine.

CH₃COCH₂COOH + FeCl₃ = Red color precipitate

If phosphates are present in the urine sample, they will get precipitated as ferric phosphate (salt).

The mixture is then heated for few minutes. If cherry or reddish color disappear, it indicates the presence of acetoacetic acid. If the color does not disappear, it indicates the absence of acetoacetic acid. Hence, the color is due to the presence of salicylates. So, Gerhardt’s test also detects salicylates in the urine.

Requirements

• Urine sample

• 10% FeCl₃

• Test Tubes

Procedure

1. A clean test tube was taken.
2. 5mL of urine sample was added to the tube.
3. Then a few drops of 10% FeCl₃ was added to the sample.
4. The test tube was kept steady for a few minutes and then observed the precipitate.
5. Then the mixture is boiled for approximately 5 minutes under around 90-95 degree Celsius and observed the mixture again.

Observation

Two samples were tested. Before boiling, a precipitate of ferric phosphate salt was seen and at the bottom of the tube a slightly light reddish color is observed in both samples. That might be acetoacetic acid. After boiling for around 5 minutes, the light reddish color did not disappear in both test tubes. There was no change of the mixture before and after boiling. So, no acetoacetic acid was present in both urine sample. The light reddish color was due to salicylates.

Result

No presence of acetoacetic acid was noticed or maybe a very negligible amount was present in both samples. 

Discussion

Ketone bodies are produced by the liver during fat metabolism. They are water soluble molecules and can be transported into tissues. In tissues, ketone bodies are converted into acetyl-CoA which enters TCA cycle and oxidized for energy.

Ketone bodies are produced in different conditions such as fasting/low food intake, starvation, carbohydrate restricted diets, prolonged exercise, untreated type 1 diabetes mellitus. We need glucose as fuel for the proper functioning of the body. During starvation or taking carbohydrate restricted diets cause low level of glucose in the body. But the brain is completely dependent on glucose. So, for the functioning of the brain, glucose must be provided. After glucose is depleted, stored glycogen starts to play the role. But glycogen stores also depleted within the first 24 hours of fasting. So, for further energy need, fatty acids come into play.  

In a normal healthy person, liver produces ketone bodies constantly and these are utilized by extra hepatic tissues. Therefore, their excretion in urine is very low and undetectable in urine tests. When ketone body synthesis rate exceeds the rate of utilization, the level of excretion in urine increases. This will cause ketosis.

In our experiment, a salt layer and light/faint reddish color at the bottom of the tube was observed. Upon heating, acetoacetic acid loses CO₂ and converts to acetone. Acetone does not react with FeCl₃. Therefore, change of color upon heating means acetoacetic acid was present. No change of color means no chance of CO₂ losing and no acetoacetic acid. So, the color was due to the presence of salicylates. That means both urine sample contained no or negligible amount of acetoacetic acid.

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