Introduction:
- Urea is the chief nitrogenous waste formed during protein metabolism in man.
- It is devised principally from the amino groups of amino acids.
- Liver is the main organ where urea is synthesized by a process called ornithine cycle.
- After urea is formed in the liver, it passes into the blood and then excreted in the urine.
- The concentration of urea in blood depends upon the relationship between urea production and urea excretion.
Normal value of blood urea
- The normal blood (serum) urea = 10-50 mg/100ml of blood.
- This value varies directly with the protein intake of the individual.
- The urea molecule contains two nitrogen atoms.
- So, the concentration of urea is expressed as blood urea nitrogen (BUN).
- The conversion of blood urea nitrogen (BUN) value to the blood urea is done by the following formula.
- Value of blood urea = BUN X 2.14
- Hence the normal BUN = 6-22mg/100ml of serum.
Clinical significance blood urea:
- Increase in BUN = The value increase in kidney disease, shock, dehydration, diabetes, acute myocardial infarction.
- Decrease in BUN = The value decreases in liver failure, impaired absorption and overhydration (increased urinary outflow).
Method for estimation of blood urea
- Blood urea can be estimated by the following method.
- Berthelot method:
- In this method to estimate blood urea, blood plasma or serum is used.
- It is based on the principle that urea is hydrolysed into carbonic acid and ammonia by enzyme urease.
- The ammonia produced is measured photometrically after its reaction with phenol in the presence of hypochlorite (Berthelot reaction).
- This blue color reaction product is determined photometrically.
- This method is widely used because of absolute specificity of enzyme urease.
Materials required:
- Buffered enzyme:
- It is prepared by missing 1ml of enzyme concentrate urease with 100ml of reagent 1 consisting of phosphate buffer.
1ml enzyme concentrate = 100ml reagent 1 =buffered enzyme.
- It is prepared by missing 1ml of enzyme concentrate urease with 100ml of reagent 1 consisting of phosphate buffer.
- Reagent 2:
- It is a ready to use buffered hypochlorite solution.
- Standard:
- This is also a ready to use solution. It consists of BUN equivalent of 37.28 mg/dl.
- The reagents are stable for 6 weeks at 2-8oC after opening.
Procedure for estimation blood urea:
- Sample to be tested (blood plasma/ serum separated out)
- Take 3 test-tubes and label them as blank, test (or sample) and standard. Add the contents in each as given.
- Blank tube- 1ml of buffered enzyme + 1ml of reagent 2.
- Test or sample tube – 1ml of buffered enzyme + 1ml of reagent 2 + 10
l of sample
- Standard tube – 1 ml of buffered enzyme + 1ml of reagent 2 + 10
l of standard solution.
- Mix the contents in each tube and incubate all three test-tubes for 10 minutes at 20-25oC.
- Measure the absorbance of the test or sample tube and the standard against the reagent blank at 600nm.