TESTING FOR KETONES Gerhards, Rotheras and Han’s Tests

TESTING FOR KETONES

Gerhardts, Rotheras and Han’s Tests

None of the commonly used methods for the detection and determination of ketone bodies in serum or urine reacts with all 3 ketone bodies. Gerhardt’s ferric chloride test reacts with acetoacetate only. Nitroprusside test are least 10 times more sensitive to acetoacetate than to acetone and give no reaction at all with beta hydroxybutyrate. So, most of the tests for ketosis essentially detect or measure acetoacetate only, so presence of ketosis may not be detected. Traditional tests for beta hydroxybutyrate are indirect; they require brief boiling of the urine to remove acetone and acetoacetate by evaporation (acetoacetate first break down spontaneously to acetone) followed by gentle oxidation of beta hydroxybutyrate to acetoacetate and acetone with peroxide, ferric ions or dichromate.  The acetoacetate thus formed may be detected with Gerhardt’s test or one of the procedures using Nitroprusside.

The three ketone bodies are acetone (2%), acetoacetic acid (20%) and 3-β-hydroxybutyrate (78%). The primary substrates for ketone body formation are free fatty acids from adipose stores. When glucose is not available ketone bodies supply the majority of the brains energy. After 3 day fast, ketone bodies provide 30% to 40% of the body’s energy requirements. In uncontrolled diabetes the low insulin concentrations result in increased lipolysis and decreased reesterification, thereby increasing plasma free fatty acids. In addition, the increased glucagon: insulin ratio enhances fatty acid oxidation in

the liver (as seen in type 1 diabetes). Increased counter-regulatory hormones also augment lipolysis and ketogenesis in adipose tissue and liver respectively. Thus increased hepatic ketone production and decreased peripheral tissue metabolism lead to acetoacetate accumulation in the blood. A major fraction is converted to β-hydroxybutyrate. In healthy people beta hydroxybutyrate and acetoacetate are present at equimolar concentrations, here acetone is minor component. In severe diabetes the ratio between beta hydroxybutyrate to acetoacetate may increase to 6: 1 owing to the presence of large concentration of NADH which favors beta hydroxybutyrate production. Measurement of ketone bodies is recommended for patients with type 1 diabetes during acute illness, stress, pregnancy or elevated blood glucose >300 mg/dL or when the patients has signs of ketoacidosis.

The term ketones refer to 3 intermediate product of fat metabolism, they are acetone acetoacitic acid and buta hydrooxybutyric acid. Ketone is found when there is excessive fat metabolism .excessive fat metabolism occurs in various situation

• Impaired ability to metabolize carbohydrate
• Inadequate carbohydrate intake
• Excessive carbohydrate loss
• Increased metabolic demand.

Method

1. Rothera's test for acetone.

2. Gerhard's test for diacetic acid

3. Lindeman's test for diacetic acid

4. Han’s method for betahydroxybutyric acid.

5. Tablet test

Principle

Nitroprusside used in this test reacts with both acetone and acetoacetic acid in presence of alkali (NH₄OH) to produce permanganent calomel red ring at the junction

Requirements

1. Test tubes
2. Rothera powder mixture

• Sodium nitroprusside
• Ammonium sulphate
• Liquior ammonia solution

Procedure

1. Transfer about  5 ml of urine to a test tube
2. Saturate with ammonium sulphate
3. add 1 crystal of sodium nitroprusside
4. Layer the liquor NH₄OH  on the side of the tube
5. Observe permanganate calomel ring at the junction of two layers.

Clinical Significance

Increased In

• Diabetes mellitus
• Propanol poisoning
• Severe starvation.
• Severe carbohydrate restriction
• Anorexia
• Fasting
• Fever
• Prolonged vomiting
• Lactic acidosis
• Salicyclate toxicity.

Note

1. In diabetes mellitus impaired ability to metabolize carbohydrate takes place. as carbohydrate cannot be used to meet the body energy need, fats are burned which leads to the presence of ketones in the urine.
2. Acetoacetic acid oxidizes rapidly to form acetone therefore test must be carried out in fresh urine specimen.
3. Individuals receiving levadopa paraldehyde pyridium and phathalein compound may produce false positive result when tested for ketonuria. Presence of salicylates give false negative result.
4. When sugar is found in urine, the urine should be tested for ketone.

See more http://slsmls.org