Urinalysis: Chemical Examination of Blood, Ketone Bodies, Urobilinogen, Bence Jones Proteins (BJP), Phenylketonuria, Porphobilinogen, Porphyrin, Calcium

Urinalysis chemical examination is a vital diagnostic tool used to detect various substances in urine, including Protein, Sugar, Bile Pigments & Bile Salts,blood, ketone bodies, urobilinogen, Bence Jones proteins (BJP), phenylketonuria, porphobilinogen, porphyrin, and calcium.

BLOOD IN URINE (HEMATURIA):

• Hematuria: Hematuria is characterized by the presence of intact RBCs in the urine. It can result from various causes, including kidney lesions, post-renal bleeding, urinary tract cancer, urinary tract infections, and more.
• Hemoglobinuria: Hemoglobinuria occurs when free hemoglobin is present in the urine. It can be associated with conditions such as intravascular hemolysis (e.g., transfusion reactions, autoimmune hemolytic anemia), severe burns, and allergic reactions.
• Myoglobinuria: Myoglobin, a muscle pigment, is found in the urine in cases of myoglobinuria. This condition can be triggered by various factors, including trauma (e.g., crush injuries, gunshot wounds, physical beatings), strenuous exercise (e.g., football, swimming), and muscle diseases.

CHEMICAL TESTS FOR BLOOD IN URINE:

1. Guaiacum Reaction:

• • Boil 5 ml of urine, allow it to cool, and add 2 drops of tincture of guaiacum.
  • Shake the mixture and create a layer with ozonic ether.
  • The presence of a blue ring after this process indicates the presence of blood.
  • Note that this test has relatively low sensitivity.

2. Reduced Phenolphthalein Test:

• • Take 3 ml of reduced phenolphthalein and add 10 drops of hydrogen peroxide (H2O2) along with 3 ml of urine.
  • Shake the mixture thoroughly.
  • The appearance of a pink color in the solution indicates the presence of blood.

Key Points:

• Hematuria can present as intact RBCs in the urine and has various potential causes, including kidney issues, post-renal bleeding, cancer in the urinary tract, and urinary tract infections.
• Hemoglobinuria involves the presence of free hemoglobin in the urine and may result from conditions like intravascular hemolysis, severe burns, and allergic reactions.
• Myoglobinuria occurs when myoglobin, a muscle pigment, is found in the urine and can be triggered by trauma, strenuous exercise, or muscle diseases.
• Chemical tests like the guaiacum reaction and the reduced phenolphthalein test can help detect the presence of blood in urine, but their sensitivity varies.

Table: Differentiation between haematuria, haemoglobinuria and myoglobinuria:

Condition/test	Microscopy	Chemical test	Saturated Ammonium sulphate precipitation test
Haematuria	Positive	Positive	Not done
Haemoglobinuria	Negative	Positive	Positive
Myoglobinuria	Negative	Positive	Negative

DETECTION OF KETONE BODIES:

• Ketone Bodies: Ketone bodies, including acetoacetic acid, β-hydroxybutyric acid, and acetone, are breakdown products of fat metabolism. Normally, they are present in urine at concentrations of up to 125 mg in 24 hours but cannot be detected by routine testing.
• Conditions with Ketone Bodies in Urine: Ketone bodies may appear in urine under various conditions, including starvation, uncontrolled diabetes mellitus, prolonged vomiting, and severe diarrhea in children, low carbohydrate diet, high-fat diet, and toxaemia of pregnancy.

Methods for Detecting Ketone Bodies:

1. Rothera Tube Test:

• • Principle: Acetoacetic acid and acetone precipitate at around 60°C and redissolve near 100°C. When urine is cooled, these precipitates reappear between 85°C and 60°C.
  • Procedure: Centrifuge fresh urine and take 10 ml of clear urine. Adjust the pH to 5.0 with 25% acetic acid. Heat the test tube slowly in a water bath with a thermometer inside. Precipitation begins at 40°C and completes at 60°C. Boil the urine to dissolve the precipitate. Upon cooling, the precipitate reappears and disappears below 40°C. The test should be confirmed by electrophoresis of concentrated urine.
    

    

2. Gerhardt’s Test (Ferric Chloride Test):

• • Principle: This test detects acetoacetic acid and is straightforward to perform. A few drops of 10% aqueous ferric chloride solution are added to 1 ml of urine.
  • Result Interpretation: The appearance of a red color indicates the presence of acetoacetic acid. The test can detect 0.5-1.0 mmol/L (5-10 mg/dl) of acetoacetic acid in urine. Gerhardt’s test may show false-positive results with salicylates, PAS, and antipyrines, as these substances are not destroyed by boiling, whereas acetoacetic acid evaporates.

DETECTION OF UROBILINOGEN:

• • Urobilinogen: Urobilinogen is a pigment produced by the bacterial decomposition of bilirubin in the intestine. It is reabsorbed and appears in urine, with trace amounts being normally present. Elevated levels may indicate increased bilirubin production and can be used to detect conditions like haemolysis and in the differential diagnosis of jaundice.

Methods for Detecting Urobilinogen:

1. Spectroscopic Examination:

• • Principle: Urine is acidified with hydrochloric acid (HCl) and examined with a spectroscope. The presence of urobilinogen is indicated by an absorption band at the junction of green and blue.

3. Bogomolow’s Test:

• • Principle: A test involving the addition of barium chloride to urine, followed by the formation of a pink or yellow color in the chloroform layer in the presence of urobilinogen.

3. Ehrlich’s Benzaldehyde Test:

• • Principle: Ehrlich’s reagent converts colorless urobilinogen into a colored compound. The appearance of a cherry red color in the presence of this reagent indicates urobilinogen.
    

    

DETECTION OF BENCE JONES PROTEINS (BJP):

• • Bence Jones Proteins: These are light chains of globulins with a molecular weight of 45,000 and are found in 40% of cases of multiple myeloma and other lymphoproliferative disorders with monoclonal dysglobulinemia. They have the potential to be eliminated through urinary excretion.

Methods for Detecting Bence Jones Proteins:

1. Heat Precipitation Test:

• • Principle: BJP precipitates at approximately 60°C and redissolve near 100°C. Upon cooling, the precipitate reappears between 85°C and 60°C.
  • Procedure: Centrifuge fresh urine and take 10 ml of clear urine. Adjust the pH to 5.0 with 25% acetic acid. Heat slowly in a water bath, and precipitation occurs at 40°C-60°C. Boil the urine to dissolve the precipitate, and upon cooling, the precipitate reappears and fades below 40°C.

2. Esbach’s Test:

• • Principle: Esbach’s reagent is used, which consists of picric acid. This reagent causes protein precipitation in urine.
  • Procedure: Dilute filtered urine with distilled water to a specific gravity between 1.006 – 1.008. Add Esbach’s reagent and mix. Allow the mixture to sit for 24 hours and then read the height of the white protein precipitate in grams per liter.

3. Pyrogallol Red Dye Test:

• • Principle: Pyrogallol red molybdate complexed with proteins at pH 2.5 produces a violet-colored compound measured at 600 nm, which is proportional to protein concentration.
  • Procedure: Perform the test using a spectrophotometer.

DETECTION OF PHENYLKETONURIA:

• • Phenylketonuria (PKU): In this disease, there is an increased concentration of phenylalanine in the blood and CSF due to a deficiency of hepatic phenylalanine hydroxylase. Phenylketones are excreted in urine and can be detected with the Ferric chloride test.

Ferric Chloride Test for Phenylketonuria:

• • Procedure: Add a few drops of 10% aqueous ferric chloride to 5 ml of fresh urine. The appearance of a greyish-green to blue-green color within 90 seconds indicates the presence of phenylketones

DETECTION OF PORPHOBILINOGEN:

• • Porphobilinogen: Porphobilinogen is detected using the Watson-Schwartz test, based on the conversion of porphobilinogen into a red-colored compound with Ehrlich’s reagent.

1. Watson-Schwartz Test for Porphobilinogen:

• • Procedure: Combine 2.5 ml of fresh urine with 2.5 ml of Ehrlich reagent, and vigorously shake the mixture for 30 seconds. The rapid development of a red coloration suggests the presence of porphobilinogen. Subsequent actions include introducing sodium acetate and pH adjustment

DETECTION OF PORPHYRIN:

• • Porphyrin: Detecting porphyrin involves adding glacial acetic acid and amyl alcohol to fresh urine and observing for salmon-pink fluorescence under ultraviolet light in the upper layer of amyl alcohol.

DETECTION OF CALCIUM IN URINE:

Calcium in Urine: The presence of calcium in urine can be screened using the Sulkowitch test. Calcium is precipitated as calcium oxalate when reacting with an oxalic acid reagent.

1. Sulkowitch Test:

Oxalic Acid Reagent Preparation:

To perform the Sulkowitch test, prepare the oxalic acid reagent by dissolving the following in 1 liter of distilled water:

• • 5 g oxalic acid
  • 5 g ammonium oxalate
  • 5 ml glacial acetic acid

Procedure:

• • Collect a 24-hour urine sample.
  • Take 5 ml of the collected urine and mix it with 5 ml of the prepared oxalic acid reagent.
  • Observe the mixture for turbidity.

Result Interpretation:

• • If no precipitate forms, it indicates the presence of normal calcium in the urine.
  • If a heavy precipitate forms, it suggests elevated levels of calcium in the urine.

The Sulkowitch test is a simple method to detect abnormal calcium levels in urine, which can be indicative of certain medical conditions or dietary factors.

Interpretation of Sulkowitch test:

No precipitate	Serum calcium <7.5 mg/100 ml
Fine precipitate	Serum calcium 7.5-11.5 mg/100 ml
Heavy precipitate	Serum calcium ≥11.5 mg/100 ml

CHLORIDE DETECTION USING FONTANA’S TEST:

• • Chloride Test: Chloride ions in urine can be detected using Fontana’s test, which involves the precipitation of chloride with silver nitrate. Excess silver nitrate then reacts with potassium chromate to produce a reddish precipitate of silver chromate.

Reagent Preparation:

• • Prepare a 20% solution of potassium chromate.
  • Prepare a 2.9% solution of silver nitrate.

Procedure:

• • Take a test tube and add 10 drops of the urine sample.
  • Add one drop of potassium chromate to the test tube.
  • Carefully add silver nitrate drop by drop until a permanent, distinct red-brown color appears.

Result Interpretation:

• • The number of drops of silver nitrate required to produce the color change is equal to the number of grams of sodium chloride per liter of urine.
  • In normal urine, the test typically requires 6-12 drops of silver nitrate.

Fontana’s test is a method for quantitatively determining the chloride concentration in urine. It is a useful diagnostic tool, particularly in cases where abnormal chloride levels may be indicative of certain medical conditions.