Rothera test used for (AIPG 2010)
|C||Bence Jones proteins|
|D||Ethers in urine|
a. The three main ketone bodies are acetone, acetoacetic acid (diacetic acid) and beta-hydroxybutyric acid. Testing for ketone bodies should be done on fresh urine or the specimen kept at 4 0C.
b. Principle : Acetone and acetoacetic acid react with sodium nitropruside in the presence of alkali to produce a purple colour.
c. Reagents : Rothera's Reagent : Dry mixture
d. Pulverize 7.5g sodium nitropruside with 200g ammonium sulphate. Store in a clean Amber bottle at 250 - 350C. Stable for 6 months.
e. Ammonia concentrated, specific gravity 0.91
f. Procedure: To about 5ml of urine taken in an 18 x 150mm glass tube, add about one teaspoon of the mixture, mix well, then add 0.5 to 1.0 ml of concentrated ammonia down to the side of the tube so that it layers on top of the urine.
g. Observe for any colour change within 30-60 seconds.
h. Result : If acetone and acetoacetic acid are present, then a purple (permanganate calomel red) colour will form at the junction of the two layers within 30-60 seconds. The result can be graded from trace to 3+ based on the intensity of the colour formed, as detailed below.
i. No change in colour - Negative
j. Pinkish ring - +
k. Red ring - ++
l. Deep purple ring - +++
m. Interpretation and quality control
n. Ketone bodies are intermediary products of fat metabolism and their presence in the blood and then in the urine are indications that the metabolism is disordered or incomplete.
o. This is associated with metabolic acidosis. This occurs in poorly controlled diabetes mellitus and also in starvation.
Normal urine does not contain methyl ketone.
p. Weak false positive reactions may occur if the urine contains L-dopa and phenyl pyruvic acid.
q. If there is suspicion of a false positive test, heat the urine in a test tube in a bunsen burner flame for one minute, allow to cool and repeat the Rothera's test. Heated urine will not give a positive Rothera's due to ketone bodies.
r. As a quality control measure, the reagent should be checked frequently using a positive control (1-2 drops of acetone is added to 5ml of urine). The use of distilled water in place of urine for negative control is recommended.