Blood urea, Formation and Excretion of Urea. Principal and Procedure of Different Methods of Urea Estimation, Reference Value, Clinical Importance. Biochemistry Unit IV. HSBTE.

The human body contains several nitrogenous products such as urea, creatinine, and uric acid. They are called non-protein nitrogen (NPN). In renal disease, an increase in the level of NPN occurs inside the blood. An increase in NPN shows renal function disorder. Total NPN estimation is not recommended in renal function disorder. Urea constitutes 50% of the total NPN. Thus urea estimation is used to detect renal function disorder. Urea concentration in blood indicates a balance between urea formation and urea excretion.

A high level of NPN in blood plasma is called azotemia. Nitrogen retention in blood plasma due to renal failure is called uremia.

BLOOD UREA: “Blood urea is the end product of protein (Amino acids) metabolism inside the human body”

FORMATION OF UREA (Urea metabolism):

The formation of urea occurs mainly in the liver. But it also takes place in the kidneys and other organs. The breakdown of amino acids in the presence of the enzyme amino acid oxidase produces ammonia and carbon dioxide. This is called oxidative deamination of amino acids or catabolism of amino acids. Ammonia is a toxic product. The liver converts ammonia and carbon dioxide into urea through the Krebs cycle (Krebs Henseleit cycle).

Kreb cycle (Krebs Henseleit cycle): The Kreb cycle utilizes two molecules of ammonia and one molecule of carbon dioxide to form one molecule of urea in each cycle. Krebs cycle takes place in the liver. It consists of the following five steps.

1. Combination of carbon dioxide and ammonia in the presence of enzyme carbamoyl phosphate synthetase to form carbamoyl phosphate.

2. Combine carbamoyl phosphate with Ornithine to form citrulline in the presence of the enzyme ornithine transcarbamoylase.

3. Combination of citrulline with aspartic acid to form argininosuccinic acid in the presence of enzyme argininosuccinic acid synthetase.

4. Conversion of arginine succinic acid to arginine in the presence of enzyme arginosuccinase.

5. Conversion of arginine to urea and ornithine in the presence of enzyme arginase. (Urea Chemical Formula: (Urea Chemical Formula: CH4N2O) )

Urea is released into blood plasma and ornithine is again utilized as in step one.

EXCRETION OF UREA:

Urea is excreted into urine from blood plasma. While some portion of blood urea is also excreted in GIT and sweat.

Urea enters blood plasma from the Krebs cycle in the liver. Blood plasma carries urea into the glomerulus of the kidney. Urea enters the proximal renal tubule through glomerular filtration. The concentration of urea in blood plasma and glomerular filtrate remains similar.

· Proximal renal tubule: Urea is not reabsorbed in the proximal tubule. However, some portion of glomerular filtrate water is reabsorbed from the proximal renal tubule. This increases the concentration of urea in the glomerular filtrate.

· Loop of Henle: Glomerular filtrate enters a loop of Henle. Some parts of the loop of Henle are permeable to urea. Thus some portion of urea is reabsorbed from the loop of Henle.

· Distal renal tubule: Distal renal tubule has low urea permeability. Some urea is reabsorbed in this segment. This decreases urea concentration in the renal tubule.

· Collecting duct: Some part of the renal tubule collecting duct has a high permeability to urea. Most of the urea is reabsorbed from this part of the collecting duct.

40% of urea is reabsorbed in the renal tubule. The rest of the urea is excreted into urine.

Principal and procedure of different methods of urea estimation:

Urea in the specimen is reported as urea mg/dl or urea nitrogen mg/dl. Urea estimation was carried out by using nitrogen-free filtrate of whole blood. The measurement of urea was based on the measurement of nitrogen in the specimen. This was referred to as blood urea nitrogen (BUN). Nowadays urea is estimated by using whole blood plasma or serum as a specimen. Thus urea should not be reported as BUN. Urea nitrogen concentration can be converted into urea concentration by multiplying urea nitrogen by 2.14.

• · Molecular weight of urea: 60

• · Molecular weight of urea nitrogen: 40

• · Urea: 2.14 x Urea nitrogen.

Methods to estimate (determine) urea or urea nitrogen: There are three methods to estimate urea or urea nitrogen in blood.

1. Diacetyl monoxime(DAM) method:

Principle: Urea reacts with diacetyl monoxime those carbazide and ferric ion in acidic media at high temperatures to form a pink-colored compound. The intensity of color is measured by a photometer to calculate urea concentration.

Reagents:

i. Reagent 1: 0.2 g/dl diacetyl monoxime solution in distilled water.

ii. Reagent 2: 40mg/dl thiosemicarbazide solution in distilled water.

iii. Reagent3:Conc.sulphuriacid60ml, orthophosphoric acid 10 ml, ferric chloride 0.1 gm in 1000ml of distilled water.

Reagent 1, reagent 2, and reagent 3 are color reagents that are mixed just before use.

iv. Urea nitrogen standard: 20mg/dl.

Procedure: Put them in tubes using a pipette as per the below details in the table.

Mix the contents of the tube properly and place them in a boiling water bath to heat them for 15 minutes. Cool them by using tap water. Measure their optical density against the blank at 520 nm (green filter).

Calculation:

Blood urea in mg/dl:= O.D. Test/O.D Std X 20.

2. Berthelot reaction method:

Principle:

• · Enzyme urease converts urea into ammonia and carbon dioxide.

• · Ammonia with phenol in the presence of hypochlorite

• · forms indophenol. Sodium nitroprusside acts as a catalyst.

• · Indophenol develops a blue color in the presence of alkali. The intensity of the blue color depends upon ammonia released from urea.

• · The intensity of the blue color is measured by a photometer at 546 nm.

Reagents:

• i. Buffered urease: urease 1000 units in phosphate buffer 100 ml, pH 6.5 to 7.0.

• ii. Phenol color reagent: Phenol 1gm, Sodium nitropruside 0.005 gm in distilled water 100ml.

• iii. Alkaline hypochlorite reagent: Sodium hydroxide 0.5gm, Sodium hypochlorite 0.042gm, distilled water 100ml

• iv. Urea standard solution: 20mg/dl

All reagents are stable at 4 degrees C for three months.

Procedure: Mix as per table and measure OD. 

Calculation:

Blood urea in mg/dl: = O.D. Test/O.D .Standard X 20.

3. U V Kinetic method :

Principle: Enzyme urease converts urea into ammonia and carbon dioxide. Alpha keto-gluterate is oxidized to glutamate in the presence of ammonia. This reaction is catalyzed by the enzyme glutamate dehydrogenase and utilizes energy from NADH. The optical density of the solution will decrease. This decrease is proportional to urea concentration. O.D. is measured by a spectrophotometer at 340 nm.

Reagents:

· Enzymes: Glutamate dehydrogenase; 900U/L, Alpha ketogluterate: 10 mMol/L, NADH: 0.25 mMol/L , Urease: KU/L.

· Diluent: Tris buffer pH 8.0

· Urea Standard: 20 mg/dl

Procedure: Measure O.D. by using a spectrophotometer.

Calculation: A “Test”/A Standard x20

Reference range (Normal range):

· Adult: 2.5 to 7.5 mmol/L or 7 to 21 mg/dl

· Slight increase in normal values after 40 years of age.

· Dangerous range: Above 100mg/dl.

· Infants: 1.4 to 5.7 mmol/L or 4 to 16 mg/dl.

Clinical importance/ clinical significance:

The amount of urea in the blood more than the normal value is called Uremia. It is mainly due to renal function disorder. The amount of NPN in the blood is more than the normal level is azotemia. It is mainly due to increased levels of urea and creatinine in the blood. Diseases related to azotemia can be divided into three groups:

• · Pre-renal condition: Blood urea level increases due to a decrease in glomerular filtration rate (GFR). The decrease in GFR is due to

  • o Decrease in renal blood flow. This happens in severe blood loss, shock, cardiac failure, dehydration

  • o Increase in protein breakdown inside the body. This happens in high fever, severe burns, stress, injury, and bleeding in soft tissues.

• · Renal condition: Blood urea increases due to renal function failure ..

• · Post renal condition: Prostate gland enlargement, kidney stone, urinary bladder tumor.

Blood urea level decreases in pregnancy, liver function disorder, and protein deficiency in diet.

Dr Pramila Singh