Introduction
- Urea is the major end product of protein metabolism in the human body.
- It is formed in the liver through the urea cycle during metabolism of amino acids.
- Ammonia produced during protein breakdown is toxic and is converted into urea for safe excretion.
- Urea contains the largest fraction of non-protein nitrogen in blood.
- After formation in the liver, urea is transported through blood to kidneys.
- Kidneys filter urea and excrete it in urine.
- Therefore serum urea level reflects both protein metabolism and renal excretory function.
- Serum urea estimation is one of the most important routine biochemical tests used to assess kidney function.
- Increased serum urea is called azotemia.
- Urea estimation is commonly performed along with creatinine for evaluation of renal status.
Principle
- Urea estimation is based on enzymatic urease–GLDH kinetic method.
- The reaction occurs in two enzymatic steps.
First Reaction
- Urea is hydrolyzed by urease enzyme in presence of water.
- Ammonia and carbon dioxide are produced.
Reaction
Urea + H₂O → 2NH₃ + CO₂
Second Reaction
- Ammonia reacts with α-ketoglutarate in presence of Glutamate Dehydrogenase (GLDH) and NADH.
- L-glutamate and NAD⁺ are formed.
Reaction
NH₃ + α-KG + NADH → L-Glutamate + NAD⁺
Principle of Measurement
- NADH absorbs light at 340 nm.
- During reaction NADH decreases continuously.
- Decrease in absorbance is proportional to urea concentration.
Specimen
Sample Type
- Serum is preferred specimen
- EDTA plasma can be used
- Heparin plasma can be used
- Urine sample can also be used
Precautions
- Use fresh non-hemolyzed sample
- Avoid ammonium heparin
- Discard contaminated specimen
Stability
Serum / Plasma
- 7 days at 20–25°C
- 7 days at 4–8°C
- 1 year at −20°C
Urine
- 2 days at 20–25°C
- 2 days at 4–8°C
- 1 month at −20°C
Urine Preparation
- Dilute urine 1:100 with distilled water
- Multiply final result by 101
Reagents
Reagent 1
- Tris buffer
- α-Ketoglutarate
- Urease
- GLDH
Reagent 2
- NADH
Standard Reagent
- Urea standard solution
Role of Reagents
- Urease – Hydrolyzes urea into ammonia
- GLDH – Catalyzes second enzymatic reaction
- NADH – Indicator molecule for kinetic measurement
Materials Required
- Test tubes
- Micropipette
- Pipette tips
- Semi-auto analyzer
- Spectrophotometer
- Cuvette
- Timer
- Water bath
- Urea reagent kit
Procedure
Two Reagent Method
| Components | Blank | Standard | Test |
|---|---|---|---|
| Reagent 1 | 1.000 ml | 1.000 ml | 1.000 ml |
| Distilled water | 0.010 ml | — | — |
| Standard | — | 0.010 ml | — |
| Sample | — | — | 0.010 ml |
First Incubation
- Mix properly
- Incubate at 37°C for 1 minute
Then Add
| Components | Blank | Standard | Test |
|---|---|---|---|
| Reagent 2 | 0.250 ml | 0.250 ml | 0.250 ml |
Reading
- Mix properly
- Read initial absorbance after 30 seconds (A1)
- Read again exactly after 1 minute (A2)
- Measure against reagent blank
- Calculate ΔA/min
Calculation
Formula
Urea (mg/dL) = (ΔA Sample / ΔA Standard) × Standard concentration
Example
- ΔA sample = 0.20
- ΔA standard = 0.25
- Standard concentration = 40 mg/dL
Calculation
- 0.20 / 0.25 × 40 = 32 mg/dL
Conversion
- mg/dL × 0.1665 = mmol/L
- Urea × 0.467 = BUN
Normal Reference Values
Serum Urea
| Group | Normal Value |
|---|---|
| Adults | 17 – 43 mg/dL |
Age and Sex Variation
| Group | Normal Value |
|---|---|
| Women < 50 years | 15 – 40 mg/dL |
| Women > 50 years | 21 – 43 mg/dL |
| Men < 50 years | 19 – 44 mg/dL |
| Men > 50 years | 18 – 55 mg/dL |
Children
| Age | Value |
|---|---|
| 1–3 years | 11 – 36 mg/dL |
| 4–13 years | 15 – 36 mg/dL |
| 14–19 years | 18 – 45 mg/dL |
Clinical Significance
- Serum urea estimation is one of the most important biochemical tests used to assess kidney function, protein metabolism, and nitrogen balance in the body.
- Urea is the major end product of protein catabolism and is produced in the liver through the urea cycle.
- Since urea is excreted mainly by kidneys, any disturbance in renal filtration immediately affects serum urea concentration.
- Serum urea level therefore reflects both hepatic production and renal excretion.
- Abnormal urea concentration is clinically important in renal disorders, liver disease, dehydration, metabolic disturbances, and systemic illnesses.
Increased Urea (Azotemia)
- Increased serum urea is called azotemia.
- It occurs when urea production increases or renal excretion decreases.
- Azotemia is one of the earliest biochemical indicators of impaired kidney function.
Renal Failure
- Renal failure is the most important cause of increased serum urea.
- In kidney disease, glomerular filtration decreases.
- Urea is retained in blood because kidneys cannot excrete it effectively.
Seen In
- acute renal failure
- chronic kidney disease
- glomerulonephritis
- nephritis
- renal obstruction
Clinical Importance
- Rising urea indicates worsening renal function
- Serial monitoring helps assess progression of kidney disease
Dehydration
- Dehydration reduces plasma volume and renal blood flow.
- Reduced filtration causes increased reabsorption of urea.
Seen In
- severe vomiting
- diarrhea
- fluid loss
- burns
Clinical Importance
- Urea rises more rapidly than creatinine in dehydration
Congestive Heart Failure
- Reduced cardiac output lowers renal perfusion.
- Decreased blood supply to kidneys causes reduced urea excretion.
Clinical Importance
- Elevated urea in heart failure suggests reduced renal circulation
Diabetes Mellitus
- Severe uncontrolled diabetes may increase serum urea.
- Dehydration and renal involvement contribute to elevation.
Clinical Importance
- Elevated urea may indicate diabetic nephropathy
High Protein Diet
- Increased protein intake increases amino acid breakdown.
- More ammonia is formed and converted into urea.
Clinical Importance
- Temporary rise may occur without renal disease
Fever and Infection
- Fever increases protein catabolism.
- Infection causes tissue breakdown and increased nitrogen metabolism.
Seen In
- severe infections
- sepsis
- prolonged fever
Gastrointestinal Bleeding
- Digested blood proteins increase urea production.
- Serum urea rises significantly.
Clinical Importance
- High urea with GI bleeding may occur without renal disease
Increased Tissue Breakdown
- Rapid tissue destruction increases protein metabolism.
Seen In
- trauma
- burns
- surgery
- severe catabolic states
Decreased Urea
- Decreased serum urea is less common than increased urea.
- It usually indicates reduced urea synthesis or dilutional state.
Severe Liver Disease
- Liver produces urea through the urea cycle.
- Severe liver damage reduces urea synthesis.
Seen In
- liver failure
- severe hepatitis
- cirrhosis
Clinical Importance
- Low urea may indicate impaired liver synthetic function
Low Protein Diet
- Reduced dietary protein decreases amino acid breakdown.
- Less ammonia is available for urea formation.
Pregnancy
- Pregnancy may lower serum urea because plasma volume increases.
- Increased renal blood flow also enhances urea excretion.
Overhydration
- Excess fluid intake dilutes serum urea concentration.
Diagnostic Importance
- Serum urea is widely used in routine renal function testing.
- It is commonly interpreted together with serum creatinine.
Clinical Uses
- assesses renal function
- evaluates protein metabolism
- monitors kidney disease
- detects dehydration
- monitors renal therapy
Blood Urea Nitrogen (BUN)
- Urea value is often converted into BUN (Blood Urea Nitrogen).
- BUN reflects nitrogen portion of urea molecule.
Clinical Importance of BUN
- widely used in renal assessment
- helps interpret renal perfusion status
- useful in ICU monitoring
Urea and Creatinine Relationship
- Urea alone is not sufficient for diagnosis.
- Creatinine must also be assessed.
Urea/Creatinine Ratio Helps Detect
- pre-renal causes
- renal causes
- post-renal causes
Monitoring Importance
- Follow-up of chronic kidney disease
- Monitoring dialysis patients
- Assessing treatment response
- Evaluating hydration status
