Biochemistry Practical 7: Urine and Serum Analysis

1. Serum Analysis

a. Albumin Quantification

• Principle: The test relies on the reaction of albumin with Bromocresol green (BCG) reagent in an acidic citrate environment, forming a blue-colored complex. The intensity of the color is directly proportional to the albumin concentration in the serum.

• Reagents:

• Bromocresol green (BCG) solution

• Citric acid solution

• Procedure:

• Prepare a standard albumin solution (40 mg/L).

• Dilute the serum sample.

• Add BCG and citric acid reagents to the serum sample and standard solution.

• Measure the absorbance at a wavelength of 590 nm.

• Results:

• Read the results from the standard curve plotted using the absorbance values of the standard solutions.

• Note:

• Normal albumin concentration in serum is 53.32 ± 4.39 g/L (representing 53.32 – 57.71% of total serum protein).

• Decreased albumin levels can occur in cases of malnutrition, cirrhosis, nephrotic syndrome, etc.

• Increased albumin levels are rare and may be associated with specific conditions.

b. Total Protein Quantification

• Principle: The test is based on the reaction of proteins with the Biuret reagent in an alkaline environment, forming a violet-pink complex. The color intensity of the complex is directly proportional to the total protein concentration in the serum.

• Reagents:

• Biuret solution (containing CuSO4 and NaOH)

• Procedure:

• Prepare a standard total protein solution (70 mg/L).

• Dilute the serum sample.

• Add the Biuret reagent to the serum sample and standard solution.

• Measure the absorbance at a wavelength of 546 nm.

• Results:

• Read the results from the standard curve plotted using the absorbance values of the standard solutions.

• Note:

• Normal total protein concentration in serum is 62 – 80 g/L.

• Decreased total protein levels can occur in cases of malnutrition, cirrhosis, nephrotic syndrome, etc.

• Increased total protein levels can occur in cases of multiple myeloma (Kahler), adrenal insufficiency, severe dehydration, etc.

2. Urine Analysis

a. Protein Determination

• Principle: The test relies on the reaction of protein with organic acid reagents (trichloroacetic acid, sulfosalicylic acid) or inorganic salts ((NH4)2SO4), forming a cloudy precipitate. The intensity of the precipitate’s cloudiness is proportional to the protein concentration in urine.

• Reagents:

• Trichloroacetic acid (TCA)

• Sulfosalicylic acid

• (NH4)2SO4

• Procedure:

• Add the reagent to the urine sample.

• Observe the appearance of the cloudy precipitate.

• Results:

• Positive protein result if a cloudy precipitate is observed.

• Note:

• Protein in urine is often precipitated by high temperature, inorganic acids, and salts.

• Proteinuria can be a sign of kidney disease.

b. Bile Pigment Determination

• Principle: This test is based on the reaction of bilirubin in urine with Fouchet reagent (containing BaCl2 and Fe3+), forming biliverdin with a green color.

• Reagents:

• Fouchet reagent (containing BaCl2 and Fe3+)

• Procedure:

• Add Fouchet reagent to the urine sample.

• Observe the appearance of green color.

• Results:

• Positive bilirubin result if a green color appears.

• Note:

• Bile pigments in urine can indicate liver disease.

c. Bile Salt Determination

• Principle: This test relies on the ability of bile salts to reduce the surface tension of water, causing precipitated sulfur powder to fall to the bottom of the test tube.

• Reagents:

• Precipitated sulfur powder

• Procedure:

• Add precipitated sulfur powder to the urine sample.

• Observe the movement of the sulfur powder.

• Results:

• Positive bile salt result if the sulfur powder falls to the bottom of the test tube.

• Note:

• Bile salts in urine can indicate liver disease.

d. Semi-Quantitative Glucose Determination

• Principle: The test is based on the reaction of glucose with Fehling’s reagent (containing CuSO4 and alkali), forming a brick-red Cu2O precipitate. The color intensity of the precipitate is proportional to the glucose concentration in urine.

• Reagents:

• Fehling’s reagent (containing CuSO4 and alkali)

• Procedure:

• Add Fehling’s reagent to the urine sample.

• Boil the mixture.

• Observe the appearance of the brick-red precipitate.

• Results:

• Negative glucose result if the solution turns green.

• Positive glucose result with a concentration below 5 g/L if a dark yellow precipitate appears.

• Positive glucose result with a concentration between 5 and 10 g/L if a brick-red precipitate appears.

• Positive glucose result with a concentration between 10 and 20 g/L if a dark yellow precipitate appears immediately upon boiling.

• Positive glucose result with a concentration above 20 g/L if a dark yellow precipitate appears immediately upon boiling.

• Note:

• Glucose in urine can be a sign of diabetes.

e. Ketone Body Determination

• Principle: The test relies on the reaction of ketone bodies with Legal’s reagent (containing sodium nitroprusside and concentrated alkali), forming a purple complex. The rate at which the purple color appears is proportional to the ketone body concentration in urine.

• Reagents:

• Legal’s reagent (containing sodium nitroprusside and concentrated alkali)

• Procedure:

• Add Legal’s reagent to the urine sample.

• Observe the appearance of purple color.

• Results:

• Positive ketone body result if purple color appears.

• Note:

• Ketone bodies in urine can be a sign of diabetes, starvation, or vomiting.

General Notes:

• Urine and serum test results are for reference only and should be interpreted in conjunction with other tests and clinical examination for accurate diagnosis.

• Sample collection and handling should follow proper technical procedures to ensure accurate results.

• Consult with your doctor regarding urine and serum testing.

This additional information is intended to enhance your understanding of Biochemistry Practical 7.