MBBS, BDS First Professional Biochemistry Practical

1. To analyze Urine for its
Abnormal Constituents.

2. Urine is said to be pathological when it contains certain
abnormal constituents in detectable amounts.
These constituents are :
• Proteins,
• Carbohydrates,
• Ketone bodies,
• Bile pigments,
• Bile salts and
• Blood.

3. Proteins

4. Normally proteins are present in urine only in traces (<150mg/day),
not detectable by routine methods.
These consist of :
• albumin,
• Tamm-Horsfall protein (mucoprotein secreted from distal tubule of
renal tract),
• some plasma proteins and
• glycoproteins.
Increased amount of proteins in urine i.e. proteinuria

5. Causes can be classified as:
Physiological / Functional:
Erect posture for longer time, strenuous exercise, pregnancy
Pathological / Organic:
Pre-renal : Infection, fever, heart disease, severe anemias,
increased intra-abdominal pressure as in ascites and tumors,
dehydration and toxemia of pregnancy.
Renal : Nephrotic Syndrome
Infection of urinary tract
Glomerulonephritis
Post-renal : Obstruction in urinary tract due to stone, stricture or tumor

6. Sometimes a peculiar protein known as Bence Jones proteins (light
chain immunoglobulins) is excreted in cases of multiple myeloma.
These proteins precipitate between 40-60ᵒC and redissolve on further
heating to 100ᵒC.
They reappear again on cooling.

7. Tests for Proteins
1. Heat coagulation test
• Fill two third of the test tube with urine solution, add 1-2 drops of chlorophenol
red indicator. The color of the solution turns pink.
• Now, heat its upper part, lower part serves as control.
• Turbidity appears in the upper part showing presence of proteins.
• If the color of solution becomes red instead of pink on adding the indicator, add
a few drops of acetic acid to reach the isoelectric pH of albumin (5.6).
• On the other hand, if color developed is yellow, add few drops of sodium
carbonate.
• Sometimes, turbidity may be due to phosphates but this dissolves by adding a
few drops of acetic acid while that due to proteins does not dissolve.

8. 2. Sulphosalicylic acid test
• Take 3 ml of urine sample and add 1-2 ml of sulphosalicylic acid
reagent.
• Shiny white ring appears confirming presence of proteins.
3. Heller’s test:
• Take 2 ml of urine sample and add 2 ml of conc. HNO₃ along the
sides of the test tube.
• A white zone of precipitated proteins appear at the junction of two
liquids.

9. Carbohydrates

10. • Normal urine contains reducing sugar in the amount of 1-1.5 g/24 hours and
less than 500 mg of glucose per 24 hours, which are not detected by routine
tests.
• Normal renal threshold for glucose is 180mg/dl.
• When this level is crossed, glucose starts appearing in urine. This condition
is known as glycosuria.

11. • Sometimes reducing sugars other than glucose may be present in urine.
These include lactose, galactose, fructose and L- xylulose.
• When renal threshold for glucose decreases, glucose is present in urine
even in the presence of normal blood sugar levels, the condition is
termed as renal glycosuria. This may be seen during pregnancy.
• In some people glucose appears in urine transiently after an excessive
ingestion of carbohydrates, the condition is termed as alimentary
glycosuria.

12. Common causes of glycosuria:
• Diabetes Mellitus
• Hyperpituitarism
• Hyperthyroidism
• Cushing syndrome
• Pheochromocytoma

13. Test for carbohydrates
Benedicts test
• Take 5 ml of Benedict’s reagent in a test tube add 8 drops of urine
solution.
• Boil it for 2 min and cool it. See the color of ppt.
• Grading of Benedict’s test:
• green colour - (<0.5 g%) - traces
• green ppt. - (0.5-1g%) - +
• yellow ppt. - ( 1-1.5 g%) - ++
• orange ppt. - (1.5-2 g%) - +++
• Brick red ppt. - (>2 g%) - ++++

14. Composition of Benedict’s reagent:
It consists of copper sulphate, sodium carbonate and sodium citrate.
CuSO₄: It furnishes Cu²⁺ in solution.
Na₂CO₃: It makes the medium alkaline.
Na citrate: It prevents the precipitation of cupric ions as cupric hydroxide by forming
a loosely bound cupric-sodium citrate complex which on dissociation gives a
continuous supply of cupric ions.

15. Reaction:
• Sugar + alkali → Enediols
• CuSO₄ → Cu⁺⁺ + SO₄⁻
• Cu⁺⁺ + Sod. Citrate → Cupric sodium citrate complex
• Enediols + Cupric sodium citrate complex → Cu⁺ + Mixture of sugar acids
• Cu⁺ + OH⁻→ CuOH (Cuprous Hydroxide)
• 2 CuOH → Cu₂O ↓ + H₂O
• Red Cuprous Oxide
• (Green, yellow and orange color appear due to combination of blue color of reagent
and different amount of red precipitates of cuprous oxide)

16. Ketone bodies
• Normally ketone bodies are not present in urine or present only
in undetectable amounts ( blood levels= 0.5-3.0 mg%).
• Ketone bodies present are:
• Acetoacetate (20%),
• Betahydoxybutyrate (78%) and
• Acetone (20%).
• These are produced in liver but utilized extrahepatically to obtain
energy.
• When blood level increases, condition is known as ketonemia
and when excreted in urine in detectable amounts, condition
termed as ketonuria.

18. Test for ketone bodies:
Rothera’s test
• Take 5 ml of urine, saturate it with solid ammonium sulphate
and add 2-3 drops of freshly prepared sodium nitroprusside
solution.
• Mix well and add liquid ammonia along the sides of the test
tube. a purple ring at the junction of the liquids indicate
presence of ketone bodies.
• It is given by acetoacetate and acetone but not by beta
hydroxybutyrate.
• If we add H₂O₂ there will be modification in the form of keto
group and the test will be given +ve by beta hydroxybutyrate.

19. Causes
• Diabetic ketoacidosis
• Starvation
• Severe vomiting
• Glycogen storage disease
• High fat diet
• Toxaemia of pregnancy

20. Blood

21. • It is normally absent in urine.
• Presence of RBCs in urine is known as Hematuria.
• It is seen in:
• Malignancy of kidney, urinary tract and bladder.
• Renal calculi
• Urinary tract infection
• Trauma to the urinary tract
• Sickle cell anemia
• Malignant hypertension

22. • Presence of free hemoglobin in urine is called hemoglobinuria.
• It is seen in intravascular hemolysis when the binding capacity of
hemoglobin is exceeded.
• Presence of myoglobin in urine is known as myoglobinuria.
• It is seen in crush injuries and muscular disorders.

23. Test for blood
Benzidine test
• Hemoglobin acts as pseudoperoxidase and gets released due to
hemolysis by acetic acid.
• H₂O₂ liberates nascent oxygen in presence of hemoglobin which
oxidises benzidine to a colored derivative.
• Take a dry test tube, add 3 mL of saturated solution of benzidine in
glacial acetic acid and 3 mL of 3% H₂O₂ solution to it.
• Now divide the contents into two parts:
• To one part add 2 mL of urine sample and to the other part add 2 mL
of distilled water to act as control.
• Appearance of bluish green color indicates presence of occult blood.