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 Introduction
Urea contributes most of the body’s non-protein
nitrogen, accounting for about 45% of the total. It is the major end-product
of protein catabolism in humans. It is synthesized in the liver, released
into blood circulation and excreted by the kidneys. Measurement of urea in
blood is a useful indicator of renal and hepatic integrity.
Principle of the method
Urea reacts directly with diacetyl
monoxime under strong acidic conditions to give a yellow
condensation product. The reaction is intensified by the presence of ferric
ions and thiosemicarbazide. The intense red colour formed is measured at 540nm/ yellow green filter.
Specimen types, collection and storage
Serum is the specimen of choice. Store samples for no
longer than 8 hours at room temperature (25-350C) and 7 days at
2-80C. For a longer duration, store in the freezer. If the samples
show evidence of bacterial contamination, do not use these for urea
estimation. Plasma could also be used for urea estimation.
Reagents
All chemicals must be Analar
grade.
 Stock
acid reagent
Dissolve 1.0g of ferric chloride hexahydrate
in 30 ml of distilled water. Add 20 ml orthophosphoric
acid and mix. Store in a brown bottle at room temperature (25-350C).Stable
for 6 months.
Mixed
acid reagent
Add slowly 100 ml of Conc. H2S04 to 400 ml distilled
water taken in a 1-litre flat-bottom conical flask kept in an icecold waterbath. Mix well and
add 0.3ml of stock acid reagent. Mix and store in a brown bottle at room
temperature (25-350C). Stable for 6 months.
Stock colour reagent – A
Dissolve 2g diacetyl monoxime in distilled water and make the volume up to 100
ml in a volumetric flask. Store in a brown bottle at room temperature (25-350C).
Stable for 6 months.
Stock colour reagent - B
Dissolve 0.5 g thiosemicarbazide
in distilled water and make up to 100 ml in a volumetric flask. Store in a
brown bottle at room temperature (25-350C). Stable for 6 months.
Mixed colour reagent
Mix 35 ml of stock colour
reagent A with 35 ml of stock colour reagent B and
make up to 500 ml with distilled water. Store in a brown bottle at room
temperature (25-350C). Stable for 6 months.
Stock
urea standard
Weigh 1.0g of analytical-grade urea and dissolve in
100ml of benzoic acid (1g/dl). Use a 100ml of volumetric flask for preparing
this. Store at room temperature (25-350C). Stable for 6 months.
Working
standard 50mg/dl
Dilute 5.0ml of stock urea standard to 100 ml with
benzoic acid. Store at room temperature (25-350C). Stable for 6
months.
Equipment, glassware and other accessories
Refer to Section A (2), Introduction to SOP
Procedure
The protocol of the procedure is described below.
Dilution
of Standards (S1-S3), Test & QC
Pipette the following into appropriately labelled 13 x 100 mm tubes
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S1
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S2
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S3
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Test
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QC
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Distilled Water (ml)
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1.9
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1.8
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1.7
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1.9
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1.9
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50 mg/dl Urea (ml)
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0.1
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0.2
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0.3
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-
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-
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Test sample /QC (ml)
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-
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-
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-
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0.1
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0.1
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Mix Well
Colour Development
The colour reagent is prepared
fresh at the time of analysis by mixing distilled water, mixed acid reagent
and mixed colour reagent in the ratio 1:1:1.
Pipette the following into another set of appropriately labelled 18 x 150 mm tubes.
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Blank
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S1
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S2
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S3
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Test
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QC
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Colour reagent (ml)
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3.1
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3.0
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3.0
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3.0
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3.0
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3.0
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Respective diluted standard ml)
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-
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0.1
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0.1
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0.1
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-
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-
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Diluted test /QC (ml)
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-
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-
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-
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-
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0.1
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0.1
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Mix all tubes well. Keep them in a boiling waterbath for 15 minutes. Remove from waterbath
and cool the tubes for 5 minutes. Set the spectrophometer/filter
photometer to zero with blank at 540nm/yellow green filter and measure the
absorbance of the other tubes.
Calculation and calibration graph
Concentration of standards:
S1
= 50 mg/dl
S2 = 100 mg/dl
S3 = 150 mg/dl
Plot the absorbance values of standards against their
respective concentrations. The measurable range with this graph is from 10 to
150 mg/dl. A calibration graph should be constructed whenever a new set of
reagents is prepared. Plot absorbance values of test/QC on the calibration
graph and read off the concentrations.
Once linearity is proved, it will be enough if S3 is set
up every time that patients’ samples are analysed
and the results calculated using the formula:
Absorbance
of test
Urea in test sample = ----------------------------- x 150 mg/dl
Absorbance of Standard
Analytical reliabilities
Refer to pages 7-9 of section 1 (General
Introduction) for the use of internal QC and interpretation of daily QC
data (for releasing patients’ results).
Since urea is one of the most common analytes
measured in a laboratory after glucose, it is recommended that an internal QC
(normal QC pool) be included with every batch of samples analysed
in the day, irrespective of the number of samples in a batch. Further, even
when a single sample is analysed as an
"emergency" sample at any time of the day or night, it is essential
to include an internal QC. From the QC results obtained for the day, mean,
standard deviation and %CV can be calculated to ensure that within-day
precision is well within the acceptable limit, i.e. 4%.
The mean value of internal QC for the day can be pooled
with the preceding 10 or 20 mean values obtained in the previous days and
between–day precision can be calculated and expressed as % CV. Ensure that
this is well within the acceptable limit, i.e. 8%.
At least once a day analyse
another QC serum from either a low QC or high QC pool.
"Assayed" QC sera with stated values (ranges)
are available from several commercial sources, viz. Boehringer
Mannheim, BioRad & Randox.
If a laboratory uses QC sera from a commercial
source, it is important that the company certifies that their QC materials
are traceable to international reference materials.
Hazardous materials
Most of the chemicals used in
this method are acids. Care should therefore be taken to avoid mouth pipetting and contact with skin.
Reference range and clinical interpretation
Serum/ Plasma Urea ……..15 – 40 mg/dl
Elevated serum urea levels may be due to pre-renal,
renal or post-renal etiology. Pre-renal causes could be cardiac related or
due to increased protein catabolism, and dehydration. Renal causes include glomerulonephritis, chronic nephritis, nephrotic syndrome and other kidney disease. Post-renal
causes include obstruction of the urinary tract.
Decreased serum urea levels could be due to pregnancy,
intravenous infusion, low antidiuretic hormone
secretion, low protein intake, severe liver diseases, inborn errors of urea
cycle and SIADH (Syndrome of inappropriate ADH secretion).
Limitations
Specimens with gross icterus
cannot be assayed as it will cause falsely elevated urea values. Do not
report results from specimen with suspected interference. Inform the
requesting physician of the problem.
References
1. Wybenga, D.R., Di Glorgio, J.&
Pileggi, V.J. (1971). Clinical Chem., 17, 891-895.
2. Seaton, B & Ali. A (1984) Med. Lab.
Sciences 41, 327 – 336.
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