Non-Protein Nitrogen: UREA

Department of Medical Technology

University of Santo Tomas
Intended Learning Outcomes

1. Apply the correct technique in the performance of NPN

compound analysis, particularly, blood urea nitrogen.
2. Analyze the laboratory results in terms of acceptability,
accuracy, precision, and reliability
3. Identify sources of error that may lead to false elevation or
decrease in the blood urea nitrogen level
4. Correlate laboratory results with clinical significance
Fate of Proteins

MRBPC UST 3
 Non-Protein Nitrogen Compounds
- Substances containing Nitrogen but are not Proteins.
- Products of catabolism of proteins and nucleic acids.
- Urea and Creatinine – most widely used to assess renal function.
- Uric Acid- diagnose disturbances in purine metabolism (i.e gouty arthritis)

Clinically important NPN Compounds

Urea 45-50%

Uric Acid 10%

Creatinine 5%

Creatine 1-2%

Ammonia 0.2%

Amino Acids 25%

MRBPC UST 4
 Urea
- Measured as BUN – Blood Urea Nitrogen
- Major metabolite product of protein catabolism
- also known as carbamide, an organic compound with chemical formula
CO(NH2)2. This amide has two –NH2 groups joined by a carbonyl (C=O)
functional group.
- Used to evaluate renal function, assess hydration status and check
nitrogen balance.

MRBPC UST 5
Analytical Methods
• Enzymatic- Urease
• GLDH – most common method
• Indicator dye – measures Urea by color change using a pH indicator
• Conductimetric – uses and electrode to measure the rate of increase
in conductivity
• IDMS – reference method, uses isotopically labelled compounds for
quanitification.

Direct Method vs. Indirect Method

• Direct
• measures a chromogen produced directly from Urea.
• method involves heating the sample with diacetyl monoxime in acid
solution. Lacks sensitivity, is time-consuming and exposure to hazard.
• Indirect – involves digesting the urea with the enzyme urease to form
ammonia and measuring the change in reaction from resultant ammonia.
MRBPC UST 6
Urease/GLDH Method

Principle: Enzymatic+ UV+ Kinetic

- 2 point kinetic measurement
- Decrease in Absorbance at 340 nm is proportional to
concentration of Urea.

MRBPC UST 7
Reagents/Materials. Instrumentation Needed
Urease/GLDH Method

MRBPC UST 8
Procedure
Urease/GLDH Method

• Prepare and label the tubes.

• Prepare the Working Reagent (Reagent1A)
• Mix 1 volume of Reagent 1 with 1 volume of Reagent 2.
• Follow the pipetting scheme below:

Question: Based on the table of reagent requirement, what is the volume of working
reagent needed? How will you prepare this? (Volume of R1 and R2)

MRBPC UST 9
 Procedure
Urease/GLDH Method

• Set-up the spectrophotometer • Proceed with measuring your

at 340 nm wavelength solutions. Start with the Blank.
Then, add the required volume
of reagent and mix the solution.
Start Incubation time.

MRBPC UST 10
Procedure
Urease/GLDH Method

Read Absorbance 1 (A1) at

27 seconds.
Continue the timer and read
Absorbance 2 (A2) at
exactly 90 seconds

MRBPC UST 11
Calculations
Urease/GLDH Method

Calculate the change in absorbance per minute. = A1- A2

Use the following formula to calculate Urea and BUN.

Question: Calculate for BUN in mg/dL of Unknown Serum. Absorbance 1 and 2 is 2.40
and 1.70 respectively. Standard’s change in absorbance is 1.0.
Answer: D in Abs (unknown) = (2.40 – 1.70); = 0.70
Concentration (Unknown) = ( 0.70 / 1.0 ) x 23.3
= 16.31 mg/dL BUN
MRBPC UST 12
 BUN vs. Urea
• A blood urea nitrogen (BUN) test measures the amount of nitrogen in your
blood that comes from the waste product Urea.

• BUN value is lower than Urea value, BUN:Urea ratio is approximately 1:2
Conversion Factors

Urea mg/dL to BUN mg/dL 0.466

BUN mg/dL to Urea mg/dL 2.14

BUN mg/dL to Urea mmol/L 0.357

Urine sample (24hr collection) should be diluted 1:20 with NSS. This dilution factor
should be used when calculating for the final concentration of urine urea in g/24hr

g/24 hr urine = g/L x Total volume in L

MRBPC UST 13
Quick Test:
Question: Convert Patient Results to BUN(mg/dL)
Patient 1: Urea = 42.92 mg/dL
Patient 2: Urea = 7.14 mmol/L

Answer:
Patient 1 = Urea mg/dL x 0.466
= 42.92 mg/dL x 0.466
= 20 mg/dL BUN

Patient 2 = Urea mmol/L x 1/0.357 Conversion Factors

= 7.14 mmol/L x 1/0.357 Urea mg/dL to BUN mg/dL 0.466
= 20 mg/dL BUN BUN mg/dL to Urea mg/dL 2.14
BUN mg/dL to Urea mmol/L 0.357

MRBPC UST 14
Reference Values
Reference Intervals
Urea Nitrogen, Adults
Plasma / Serum 6- 20 mg/dL 2.1 – 7.1 mmol Urea/day
Urine, 24 hr 12-20 g/24hr 0.43 – 0.71 mol Urea/day

MRBPC UST 15
 Specimen Requirements
• Serum or Plasma, Timed Urine specimen (24 hour urine collection)

Effect of a single protein-

Sodium Citrate and
containing meal is
Sodium fluoride inhibit
minimal. Fasting is not
urease.
required.

Timed urine specimen

Hemolyzed samples
should be refrigerated
should be avoided for
during collection period.
potential interference.
Urea is susceptible to
bacterial decomposition.

MRBPC UST 16
 Clinical Significance

Azotemia – elevated Urea in the

blood.

Uremia/ Uremic Syndrome –

Very high plasma Urea with renal
failure.

MRBPC UST 17
Hypothetical Readings
To be used for LabSheet Ex. 9.1

MRBPC UST 18
Hypothetical Readings
To be used for LabSheet Ex. 9.1

Reference Intervals
Urea Nitrogen, Adults
Plasma / Serum 6- 20 mg/dL 2.1 – 7.1 mmol Urea/day
Urine, 24 hr 12-20 g/24hr 0.43 – 0.71 mol Urea/day

MRBPC UST 19
MRBPC UST 20