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.
o Low carbohydrate diet.
o Pregnancy or lactation.
o Eclampsia.
o Fever.
o Hyperthyroidism.
o Prolonged vomiting and diarrhea.
o Patients in the hospital may have
ketonuria—roughly 15% show ketonuria
in hospitalized patients without diabetes.
o Ketonuria may develop after anesthesia
with ether and chloroform.
o 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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