PHR 375 Practical Biochemistry

1. Pharos University in Alexandria Faculty of Pharmacy PHR 375 Practical Biochemistry

2. Practical “Week 3” Colorimetric estimation of urea in plasma

3. Introduction Urea is a nitrogenous excretory waste product produced by the liver during the urea cycle; it results from protein catabolism, where amino acids are metabolised by a process called deamination which results in the formation of ammonia.

4. Introduction Since ammonia is toxic to cells, the body gets rid of it by converting it into the water soluble compound, urea, through the urea cycle that occurs in the liver.

5. Introduction As urea is produced, it leaves the liver to all body parts and is removed by the kidneys and excreted in urine. If the kidney function is impaired, urea level rises in blood. A simple test is carried out to test the renal function by the determination of urea or blood urea nitrogen (BUN), which is a measure of the amount of nitrogen in the blood that comes from urea.

6. Aim of practical session The aim of this practical session is to: • Obtain a simplified knowledge about urea and the clinical significance of altered urea levels in plasma. • Determine the level of urea in the provided plasma sample of different individuals and comment on the case.

7. Normal Value The normal value of plasma urea is 15 - 39 mg/dL, which is equivalent to 7- 18 mg/dL BUN.

8. Clinical significance • High plasma urea (or BUN) values occur due to: • Direct damage of the kidneys such as glomerulonephritis or pylonephritis. • Diseases that directly damage kidneys such as diabetes or high blood pressure. • Decreased blood flow to the kidneys caused by dehydration, shock or heart failure.

9. Clinical significance • Obstruction of urinary tract by a kidney stone or tumour. • Bleeding in the gastrointestinal tract (e.g. from ulcers). • Eating unusually-high protein foods. • High stress on the body, such as from a high fever. • Medications such as aspirin, diuretics, tetracyclines and aminoglycosides.

10. Clinical significance • Low plasma urea (or BUN) values occur due to: • A very low-protein diet. • Malnutrition. • Severe liver damage. • Overhydration due to drinking excessive amounts of liquid. • Urea levels may normally be low in the third trimester of pregnancy. • Syndrome of inappropriate antidiuretic hormone secretion (SIADH).

11. Modified Berthelot Enzymatic method This method utilizes the principle of urea hydrolysis. Urea is hydrolyzed in the presence of urease to carbon dioxide and ammonium ions. In alkaline medium, ammonium ions reacts with hypochlorite and salicylate to form dicarboxyindophenol, a coloured compound. The reaction is catalyzed by sodium nitroprusside. The intensity of the color produced is proportional to the concentration of urea and can be measured spectrophotometrically at max 600 nm. Estimation of Urea in plasma

12. In a clean dry test tube, using a proper automatic pipette, add 20 µl of distilled water (blank) or standard urea solution (standard) or plasma (test), then with another proper automatic pipette add 0.5 ml of Reagent (A). Practical Reagent (A) is composed of: Sodium salicylate, sodium nitroprusside, urease enzyme and phosphate buffer at pH 6.9.

13. Mix thoroughly and incubate the test tubes for 5 min at 37 ºC using a water bath. Using another automatic pipette, add 0.5 ml of Reagent (B). Practical Reagent (B) is composed of sodium hypochlorite and sodium hydroxide. • Mix thoroughly and incubate the test tubes for 5 min at 37 ºC using a water bath. Remove the test tubes from the W.B.

14. Measure the absorbance of the final color of the test and standard against blank at max 600nm. Calculate the concentration of urea in the provided sample using the absorbance reading of standard urea and applying the following equation: Practical Ctest = Cstd  Atest / Astd Comment on the case.

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