Estimation of blood urea by Urease method

Introduction
A healthy human adult degrades approximately 200g of protein daily (mostly from muscle) to
yield amino acids. 75 to 80% of these amino acids are reutilized for protein synthesis. The
remaining are catabolized in the liver, releasing 5-15g of nitrogen in the form of ammonia.
As ammonia is toxic and it is converted to urea. Urea is a non-protein nitrogen containing
compound, which forms 55-90% of urine nitrogen and is synthesized in the liver, through the
urea cycle (also called Krebs-Hensleit cycle/ ornithine cycle)
Normal Serum Urea Level 20 to 40 mg/dl.
Estimation of urea is done by enzymatic method using urease and glutamate dehydrogenase.
Serum urea is sometimes expressed in terms of its nitrogen, because nitrogenous substances
were analyzed by Kjeldahl method. Such expression of Urea-N or blood urea nitrogen (BUN)
is very common in USA. Molecular weight of urea is 60 and each g.mol of urea contains 28 g
of nitrogen.
Thus a serum concentration of 28 mg/dl of BUN is equivalent to 60 mg/dl of urea or 10
mmol/L of urea. BUN can be converted into urea by multiplying the figure by 2.14.

Interpretation of Blood Urea Value

Urea is the end-product of protein metabolism
The serum concentration of urea generally increases as the age advances.
The lower range is usually seen in young adults and the upper limit is normal for elderly
people.
Therefore a value of 40 mg/dl in a patient of 25 years may be considered as suspicious, while
the same value in a person of 60 years can be considered as perfectly normal.
Renal Diseases
Serum urea is increased in all forms of kidney diseases. In acute glomerulonephritis values
may be as high as 300 mg/dl.
In early stages of nephrosis, serum urea may be normal, but in late stages serum urea
increases along with decreasing renal functions.
In malignant hypertension and in chronic pyelonephritis, the values may reach very high
levels.

Biological reference interval Blood Urea : 15-45mg/dl

Physiological variation
• More in males
• Increase with age and high protein diet
• Decrease in pregnancy (due to hemodilution)

Clinical significance
Increase in blood urea is seen in
Pre-renal causes
Dehydration
Shock
Severe burns
Haemorrhage
Renal causes
Acute and chronic glomerulonephritis
Later stages of nephrosis
Polycystic kidney
Malignant hypertension
Hydronephrosis

Post renal causes

Enlargement of the prostate gland
Stones in urinary tract and bladder
Tumours of the bladder
Decrease in blood urea is seen in
Severe liver disease (viral hepatitis)
Decreased protein intake
Aim: To determine concentration of blood urea by Urease- GLDH(glutamate
dehydrogenase) method.
Principle

It is a kinetic method. In the presence of Urease enzyme, urea gives ammonium and
bicarbonate. The ammonium ion in the presence of GLDH enzyme converts 2-oxoglutarate
into glutamate and NAD+ is released. The rate of decrease in absorbance per minute is
measured at 340nm.

Reagent composition
R1 : TRIS buffer pH 7.8 120mmol/L
α- ketoglutare 7mmol/L
ADP 0.6mmol/L
Urease >6kU/L
GLDH >1kU/L
R2: NADH 0.25mmol/L
Standard 50mg/dl

Reagent preparation
working reagent : mix 4 parts of R1 + 1 part of R2
Specimen : serum
Procedure: Take 2 test tubes and label as standard (S) and Test (T). Add 10 microlitre of
standard solutionin S test tube, add 10microlitre of test sample in T test tube. Add
1000microlitres of working reagent in both S and T test tubes. Mix well, incubate for
0
30seconds at 37 C and read the absorbance at 340nm.
switch on analyser, select KFT from main menu, select blood urea, select wash and insert
probe in distilled water, select standard on display, insert probe in standard solution once
aspirated wait for few seconds, the absorbance will be displayed, make a note of it. Perform
wash, select test sample insert probe in T tube, once test sample is aspirated wait for few
seconds absorbance will be displayed, make a note of it. Using the following formula,
calculate blood urea level
Calculation :
Blood urea (mg/dl) = absorbance of Test/ absorbance of sample × conc. of standard
Report : The concentration of urea in the given blood sample is ------mg/dl
Other methods of estimation:
Diacetyl Monoaxime method
Urea reacts with DAM in hot acid medium in presence of thiosemicarbazide to give pink
colored complex. The intensity of it is directly proportional to the conc. of urea in the sample,
this is measured colorimetrically at 540nm.
Phenol Hydrochloride method
NH3 reacts with phenol sodium hypochloride to form glutamic acid. The conc. of NAD
formed is directly proportional to the conc. of urea.
Glutamine dehydrogenase method
Blood Urea Nitrogen( BUN)
It is the amount of Nitrogen in blood urea
BUN= blood urea conc. × gram molecular weight of nitrogen/ gram molecular weight of urea
BUN= conc. of urea × 0.46
Normal value of BUN = 7-18mg/dl
Serum creatinine and blood urea nitrogen concentrations are usually elevated and are used as
an estimate of the degree of renal damage or, more appropriately, as an estimate of remaining
functional renal capacity.

Causes for Increased Blood Urea

1. Pre-renal conditions
Dehydration: Severe vomiting, intestinal obstruction, diarrhea
Diabetic coma and severe burns
Fever and severe infections

2. Renal diseases
i. Acute glomerulonephritis
ii. Nephrosis
iii. Malignant hypertension
iv. Chronic pyelonephritis

3. Post-renal causes
Stones in the urinary tract
Enlarged prostate
Tumors of bladder

4. Medications
ACE inhibitors
Acetaminophen
Aminoglycosides
Amphotericin B
Diuretics
NSAIDs
Importance of Urea Clearance
The urea clearance is less than GFR, because urea is partially reabsorbed. Urea clearance is
the number of ml of blood which contains the urea excreted in a minute by kidneys.
Procedure
Allow the patient to have a normal breakfast. At 9 am give a cup of water and the patient is
instructed tovoid the bladder, and urine is discarded. At 10 am
bladder is completely emptied and the volume of urine is measured and the urine urea is
estimated.
A blood sample is taken and blood urea is also estimated.
Maximum Urea Clearance
The urea clearance is calculated by the formula
U x V/P
where U = mg of urea per ml of urine; P = mg of urea per ml of plasma and V = ml of urine
excreted per minute. This is called maximum urea clearance and the normal value is found to
be 75 ml/minute.
Standard Urea Clearance
But the clearance value is decreased when V, the volume of urine, is less than 2 ml/minute.
Then it is called standard urea clearance, where the normal value is found to be 54 ml/minute,
and is calculated as:
Ux√V
---------
P

Interpretation of Urea Clearance Value:

i. If the value is below 75% of the normal, it is considered to be abnormal. The values fall
progressively with failing renal function.
ii. The clearance value may be abnormal even though the plasma urea values are within
normal limits. The plasma urea values will start to rise only when the clearance value falls
below 50% of the normal.
iii. Urea is normally reabsorbed from renal tubules and therefore tubular function also affects
urea clearance. Hence, creatinine
clearance test is more preferred.
iv. Urea is freely filtered by the glomerulus and passively reabsorbed in both PCT and DCT.
Decreased Blood Urea
Urea concentration in serum may be low in late pregnancy, in starvation, in diet grossly
deficient in proteins and in hepatic failure.