Ketone body examination in urine by Rother's Test

A ketones in urine test measures ketone levels in your urine. Normally, the cells in your body use glucose (sugar) from your blood for energy. If your cells can't get enough glucose, your body breaks down fat for energy instead. This produces an acid called ketones, which can build up in your blood and urine.

1. SCHOOL OF PARAMEDICS AND ALLIED HEALTH SCIENCES, BBSR Presented By: ARPITA MOHAPATRA Programme: Master of Science Branch: Applied & Clinical Microbiology Guided By: Asst. Prof. Sonalika Mahapatra

2. CONTENTS Urine and its Composition Ketone bodies in urine Ketone bodies: Types Terminologies related to Ketone bodies Clinical significance of Ketone Bodies Detection of Ketone Bodies- Rothera’s Test 2024 Clinical pathology 2

3. URINE AND ITS COMPOSITION Urine: the waste product of kidney, a blood filtered waste product, compositing of 96% water, 2% urea, 2% other ions (Ammonia, sodium, Potassium, Chloride, etc.) Ph- Slightly acidic about 6.2 The normal constituents excreted in urine are urea, chloride, sodium, potassium, creatinine, dissolved ions, organic and inorganic compounds. Abnormally, are the excretion of detectable amount of glucose, ketone bodies, bile salt, etc. 2024 CLINICAL PATHOLOGY 3

4. KETONE BODIES IN URINE Abnormal constituent of urine- Ketone bodies When food is digested, the body turns carbohydrates in glucose that enters the blood stream. The body needs glucose as a fuel to perform activities. However, glucose has to be delivered, it does not automatically route itself to body site requiring fuel insulin, a hormone secreted by the pancreas carry out the task, delivering glucose to the cells throughout body cells and tissues. When there is not enough insulin in body is unable to use sugar for energy and body start burn fat for energy, Ketone bodies are the byproduct for this process. 2024 CLINICAL PATHOLOGY 4

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6. KETONE BODIES: TYPES Acetone Present in 2% True ketone body Acetoacetic acid Present in 20% True ketone body Betahydroxybutyrate Present in 78% 2024 CLINICAL PATHOLOGY 6 Ketone bodies consists chemically of three substances:

7. TERMINOLOGIES 2024 CLINICAL PATHOLOGY 7 Normal Plasma level of ketone bodies- <<1mg/dl Normal urinary level of ketone bodies- <<3mg/dl Ketonemia - The presence of an abnormally high concentration of ketone bodies in blood. Ketonuria - A condition in which abnormally high amounts of ketone bodies are present in urine. Ketonuria also known as ketoaciduria. Ketoacidosis- serious diabetes complications, where body produces excess blood acids (ketones) i.e., Diabetic ketoacidosis, diabetic acidosis.

8. CLINICAL SIGNIFICANCE OF ketone bodies The ketones bodies are seen in case of: Diabetes mellitus Starvation Fasting Fever Low carbohydrate diet Increased protein diet Prolonged vomiting Pregnancy Anorexia 2024 CLINICAL PATHOLOGY 8

9. DETECTION OF KETONE BODIES IN URINE ROTHERA’S TEST is performed to detect the presence of ketone bodies in urine. Principle: Acetoacetic acid and acetone reacts with sodium nitroprusside in alkaline solution to form a purple colored complex. Rothera’s test is sensitive to 5mg/dl of acetoacetic acid and 10mg/dl of acetone in urine. Either we can use commercially made Rothera’s powder compositing of 0.75gms of Sodium Nitroprusside and 20gms of Ammonium sulphate. Else single compounds could also be taken prior to use. 2024 CLINICAL PATHOLOGY 9

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11. 2024 CLINICAL PATHOLOGY 11 Requirements: Test tubes, Sodium Nitroprusside, Ammonium sulphate, Liquor Ammonia, Dropper. Procedure: Transfer about 5 ml of urine to a test tube. To it ammonium sulphate was added in pinch for saturation. The saturation process tend to bring up all the ketone bodies to the surface of the urine Saturate till deposition comes on the bottom of the test tube To it a crystal of Sodium Nitroprusside was added and mixed. To the mixture then Liquor ammonia was added (8 drops) alongside of the wall. Result and Interpretation: Immediate formation of purple permanganate coloured ring at the interface- Ketone bodies “+ve” The intensity of the colour formed was graded +, ++, +++.

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13. Quantitative detection Quantitative detection: Blood test Detection of ketones in blood. 2024 CLINICAL PATHOLOGY 13

14. UPDATE ON MEASURING KETONES Recently technology has announced for measuring of beta-hydroxybutyrate in interstitial fluid. Understanding the potential mechanism of SGLT 2 which may be involved in the promotion of ketosis and ketoacidosis in Patients. Understanding the mechanism of T1D & T2D patients. Differentiation on the terminologies- Ketogenesis, Ketosis, Ketoacidosis and Diabetic Ketoacidosis 2024 CLINICAL PATHOLOGY 14

15. 2024 CLINICAL PATHOLOGY 15 REFERENCES Giri, D. (2018, November 5). Rothera's Test for Ketone Bodies: Principle, Procedure and Clinical significance Retrieved from LABORATORY TESTS.org . : https://laboratorytests.org/rotheras-test/ Godkar, P. B., & Godkar, D. P. (2018). DETERMINATION OF KETONES. In P. B. Godkar, & D. P. Godkar, TEXTBOOK OF MEDICAL LABORATORY TECHNOLOGY (3 ed., Vol. 2, pp. 1436, 1437). Mumbai: Bhalani Publishing Hose. ISBN 9789381496190 Jingtong , H., Yeung, A., & Bergenstal , R. (1.02.2023). Update on Measuring Ketones. San Francisco: UC San Francisco Previously Published work. doi:10.1177/19322968231152236

16. 2024 CLINICAL PATHOLOGY 16 THANK YOU