Test for ketone bodies, bile salts and.pptx

Test for pathological urine-II
Test for ketone bodies, bile salts and blood in pathological urine sample. Brief overview of the parameters.

1. Test for ketone bodies, bile salts and blood in urine Prof (Dr) Viyatprajna Acharya, MD, PhD

2. Ketone Bodies Ketone bodies are produced when glucose is not available as fuel source, e.g. Starvation & DM

3. KETOGENESIS SITE: Liver ORGANELLE: Mitochondria Acetoacetate also formed from degradation of carbon skeleton of ketogenic amino acids like Leu, Lys, Phe , Tyr Normal level: 0.2 mmol/l or <1mg/dl in well-fed state Renal threshold- 70mg/dl

4. KETOLYSIS Utilized in extra hepatic tissues Cardiac Mm. and renal cortex – prefer Sk. Mm.& brain – Alternate source of energy Before utilization, acetoacetate is activated to acetoacetyl CoA Oxidation of KB can go up to the level of 12mmol/l Ketonaemia is due to ↑production of KB rather than deficiency in peripheral utilization

5. Causes of ketosis Primary substrate for KB formation is FFA from adipose stores Brain- utilises KB after glucose, can’t use FFA for energy After 3 days fasting, KB takes over – 30-40% body’s energy expenditure

6. Uncontrolled DM; FBS > 300mg/dl- possibility of ketosis 1. Insulin deficiency ↓ Increased lipolysis and decreased re-esterification 2. Increased glucagon:insulin ratio favours FA oxidation in the liver 3. Increased counter-regulatory hormones– augment lipolysis and ketogenesis All put together- increased ketogenesis and accumulation in periphery ↓ ketosis

7. KB are found in Diabetic patients. Starvation. Fasting. High protein diet. Alcoholism. Salicylates poisoning. Isopropanol intake. Acute febrile illness (especially in infants and children). Renal Glycosuria. Glycogen storage disease (Von Gierke’s disease). Anorexia. Low carbohydrate diet. Pregnancy or lactation. Eclampsia. Fever. Hyperthyroidism. Prolonged vomiting and diarrhea . Patients in the hospital may have ketonuria—roughly 15% show ketonuria in hospitalized patients without diabetes. Ketonuria may develop after anesthesia with ether and chloroform. Children are more prone to develop ketonuria and ketosis.

8. Outcome Electrolyte imbalance. Dehydration. If not corrected, then it leads to acidosis, coma, and ultimately death. Ketones are present in the urine when a threshold level of ketones exceeds the blood’s normal level. Ketonemia – upto 20mg/dl Ketonuria- when 70mg/dl is reached

9. Rothera’s test Principle- Nitroprusside in alkaline solution reacts with a ketone group to give a purple coloured complex. This given by acetone and acetoacetic acid but not by β-hydroxybutyrate. Procedure- Saturate 5ml of urine with solid ammonium sulfate. To it add 0.5ml of freshly prepared 5% sodium nitroprusside solution. Mix well and add 1ml of liquid ammonia along the sides of the tube. A purple ring is formed at the interface

10. Gerdhardt’s ferric chloride test for acetoacetate Principle- Acetoacetic acid gives a red colour with ferric chloride. Procedure- To 5 ml of urine add 10% ferric chloride drop by drop. If phosphates are present then a precipitate of ferric phosphate is formed. It has to be filtered off and to the filtrate add further ferric chloride. A wine red colour develops. This test is given by acetoacetate alone. Similar colour is given by salicylates, phenol and antipyrine. This can be differentiated by heating. On heating, acetoacetate gets evaporated to acetone and the colour disappears but for other agents it remains the same.

11. False positives and negatives Some of the drugs give false-positive results Phenothiazine. Ether and chloroform. Metformin. Captopril. Insulin. Levodopa. Penicillamine. Isopropyl alcohol. Valproic acid. Aminosalicylic acid. The false-negative result is seen in the following: When urine is kept at room temperature for a long time due to the loss of ketones in the air. Aspirin. Phenazopyridine

12. Test for Bile salts

13. Bile acids & salts are synthesized in the liver from cholesterol- Emulsification of fats and aid in absorption Primary bile acids- Cholic acid & Chenodeoxycholic acid Bile salts are formed by conjugation of primary bile acids with Glycine and Taurine- Na & K salts of glycocholate and Taurocholate and glycochenodeoxycholate and taurochenodeoxycholate Secondary bile acids are formed by deconjugation of primary bile acids by intestinal bacteria- Cholic acid →Deoxycholic acid Chenodeoxycholic acid → Lithocholic acid

14. Hay’s test Principle- Bile salts lower the surface tension thereby sinking the sulphur powder. Procedure- Take 2 test tubes, one containing 10ml of urine and the other containing 10 ml of water. A pinch of sulfur powder is sprinkled over both. Sulphur will start to sink in the tube containing bile salt.

15. Presence of bile salts in urine Obstructive jaundice- +++ Hepatocellular jaundice- +

16. Benzidine test for blood

17. Hematuria is more commonly encountered Principle- Peroxidase liberated from damaged RBCs act on hydrogen peroxide to liberate nascent oxygen which oxidizes Benzidine to a coloured derivative. Procedure- Take a pinch of Benzidine powder and shake it well with 3ml of glacial acetic acid. Add 1ml of hydrogen peroxide and mix well. Suspend 1 ml of urine carefully along the sides of the tube. A blue or green colour at the top indicates blood in urine.

18. Causes of Hematuria and hemoglobinuria Gross Haematuria :  Renal stones  Glomerulonephritis  Trauma to kidney  Tuberculosis Microscopic Haematuria: o Malignant hypertension o Sickle cell anaemia o Polycystic kidney disease Causes of haemoglobinuria: • Malaria • G-6 PD deficiency • Haemolytic uraemic syndrome • Unstable haemoglobin diseases • Incompatible blood transfusion. Black water fever (Falciparum malaria)

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