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UREA NITROGEN RATE (UV) PROCEDURE
Intended for the Quantitative Determination of Urea Nitrogen in Serum
SUMMARY AND EXPLANATION
Urea represents the end product of body protein catabolism and is cleared through the
kidneys by glomerular filtration. Urea Nitrogen is the most frequently performed test for
renal function, and along with the creatinine test yields differential diagnosis of
hyperuremia. Among the causes of elevated urea are acute glomerulonephritis, chronic
nephritis, polycystic kidney, nephrosclerosis, tubular nephrosis, and obstruction of the
urinary tract (1). Previously, urea has been determined by the direct method where urea
condenses with diacetyl to form a chromagen and an indirect method where ammonia is
measured as a product of urea acting upon urease. The liberated ammonia is measured by
the Berthelot reaction (2). Talke and Schubert introduced a totally enzymatic procedure
in 1965 utilizing urease and glutamate dehydrogenase (3). The procedure presented is a
modification of their method.
PRINCIPLE
Urea is hydrolyzed by urease to produce ammonia. The liberated ammonia reacts with 2-oxoglutarate in the presence of NADH to yield glutamate. An equimolar quantity of
NADH undergoes oxidation during the reaction resulting in a decrease in absorbance at
340 nm, that is directly proportional to urea nitrogen concentration.
Urea + H2O urease 2NH3 + CO2
NH3 + a-Ketoglutarate + NADH + H GD L-glutamate + NAD + H20
REAGENTS: FOR IN-VITRO DIAGNOSTIC USE
Reagent Set Cat. No. 4060 provides:
UREASE REAGENT - (Cat. No. 4061)
REACTIVE INGREDIENTS:
Glutamate Dehydrogenase - 5000 u/L; a-Ketoglutarate - 4.0 mM; NADH - 0.30 mM;
Urease - 1500 u/L; Buffer to pH = 8.2, stabilizers and fillers added.
PRECAUTIONS:
Causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash
with large amounts of water.
STORAGE AND STABILITY:
Store at 2 - 8ºC. Stable until expiration date if sealed tightly. Reconstitute with amount of
ammonia-free distilled water stated on vial label. Swirl gently to dissolve. DO NOT
SHAKE. Reconstituted reagent is stable for 2 days at room temperature and for 30 days
at 2 - 8ºC.
DETERIORATION:
The reagent should be a dry, white powder. Reconstituted reagent should be a clear,
colorless solution. Turbidity indicates contamination, and the reagent should not be used.
Also, if the reconstituted reagent has a reagent blank absorbance of less than 1.0 when
read at 340 nm against distilled water, it should not be used.
UREA NITROGEN CALIBRATOR - (Cat. No. 4062)
REACTIVE INGREDIENTS:
30 mg/dL urea nitrogen. Preservative added.
PRECAUTIONS:
Causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash
with large amounts of water.
STORAGE AND STABILITY:
Store at 2 - 8ºC. Stable until expiration date if sealed tightly.
DETERIORATION:
The calibrator should be a clear, colorless solution. Turbidity would indicate
deterioration.
INSTRUMENTS
Use a spectrophotometer or colorimeter calibrated at 340 nm.
SPECIMEN COLLECTION
PRECAUTIONS:
1. Fresh, unhemolyzed serum is recommended.
2. Plasma containing anticoagulants should not be used.
SAMPLE STORAGE:
Urea in serum is reported to be stable for 8 hours at room temperature or for up to 72
hours when stored at 2 - 8ºC.
ADDITIVES:
No special additives or preservatives are needed.
INTERFERING SUBSTANCES:
1. Plasma collected in anticoagulant containing either ammonium or flouride salts
cannot be used.
2. Urine cannot be used as a sample due to interference by amounts of preformed ammonia that may be present.
3. Avoid ammonia contamination of reagents and glassware.
4. Ammonia free water must be used for reconstitution of Urease Reagent.
5. Young et al, (4) have comprehensive reviews of drug effects of urea nitrogen
levels.
PROCEDURE
MATERIALS PROVIDED:
UREA NITROGEN RATE REAGENT (Cat. No. 4061) and UREA NITROGEN
CALIBRATOR (Cat. No. 4062).
MATERIALS REQUIRED BUT NOT PROVIDED:
1. 1.0 mL and 0.01 mL pipettors
2. Test tubes, rack and timer
3. Instrument capable of reading at 340 nm
REACTION CONDITIONS:
Wavelength 340 nm
Reaction Type Kinetic
Incubation Temperature Ambient
Preincubation Time 30 seconds
Reaction Time 1 minute
Sample Volume 0.01 mL
Reagent Volume 1.0 mL
Final Volume 1.01 mL
Low Normal 7 mg/dL
High Normal 18 mg/dL
Calibrator Value 30 mg/dL
PREPARATION OF WORKING REAGENT:
Reconstitute the reagent with the amount of ammonia-free distilled water specified on
the vial label. DO NOT SHAKE.
AUTOMATED PROCEDURE:
Refer to specific instrument application for instructions.
MANUAL PROCEDURE:
1. Pipet 1.0 mL reconstituted reagent into tubes labeled Calibrator, Control, Sample 1, etc. Allow reagent to come to room temperature before use.
2. Adjust instrument to zero absorbance at 340 nm using distilled water.
3. Place 0.01 (10 uL) specimen into appropriate tube, mix and immediately place in
the instrument. Allow to stand for 30 seconds.
4. After 30 seconds, record the absorbance (A1).
5. After exactly 1 minute, read and record the absorbance (A2).
6. Determine the absorbance change between the two readings: (A1 - A2).
PROCEDURAL NOTE - For instruments requiring a total volume 1.0 mL to read,
increase Reagent Volume to 2.0 mL and Sample Volume to 0.02 mL and perform test as
directed in MANUAL PROCEDURE.
STABILITY OF FINAL REACTION PRODUCT:
The reaction is ongoing, the timing of the readings must be accurate.
CALIBRATION:
It is not necessary to perform a calibration curve with this procedure since the reaction is
linear in the range of 0 - 80 mg/dL. However, a Calibrator must be determined with each
set of Unknowns assayed. Use UREA NITROGEN CALIBRATOR (Cat. No. 4062)
which is provided in the reagent set for this purpose.
QUALITY CONTROL:
The reliability of test results should be monitored routinely using suitable quality control
materials (normal and abnormal) analyzed in the same manner used for Unknowns.
EAGLE DIAGNOSTICS offers CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of
freshly prepared control sera should be thoroughly investigated before patient values are
reported.
CALCULATION OF RESULTS
The following equation is used to determine Unknown concentrations:
Unknown (mg/dL) =
(A1 - A2) Unk. Abs.
-------------------------- X Conc. of Cal.(mg/dL)
(A1 - A2) Cal. Abs.
EXAMPLE:
A 30 mg/dL Calibrator had its first absorbance reading (A1) = 1.50 and the absorbance
reading after 1 minute (A2) = 1.05, the unknown absorbance reading (A1) = 1.50 and the
absorbance reading after 1 minute (A2) = 0.94, the concentration of the unknown is:
1.50 - 0.94
---------------- X 30 mg/dL = 37 mg/dL
1.50 - 1.05
LIMITATIONS
1. Specimens with values greater than 80 mg/dL should be diluted 1:1 with ammonia-free deionized or distilled water and reassayed multiplying the result by two.
2. Ammonia-free deionized or distilled water must be used for reconstitution of
reagents.
EXPECTED VALUES (1)
7 - 18 mg/dL
The ranges represents the 95% confidence intervals from a clinically normal population.
It is recommended that each laboratory establish its own range of expected values.
PERFORMANCE CHARACTERISTICS
LINEARITY:
This method is linear to 80 mg/dL.
PRECISION:
Normal and abnormal control sera were assayed 20 times each for within run precision
and for 10 working days to establish run to run precision.
WITHIN RUN
MEAN / ST. DEV. / %CV
Normal 15.0 / 0.65 / 5.1
Abnormal 47.0 / 0.95 / 1.9
RUN TO RUN
Normal 15.0 / 0.45 / 3.9
Abnormal 48.0 / 1.10 / 2.2
SPECIFICITY:
A comparison of this UREA NITROGEN RATE (UV) PROCEDURE with another
widely used commercial method showed a 99.2% correlation with samples in the normal
and abnormal range.
SENSITIVITY:
This procedure has a sensitivity of 0.03 mg/dL per 0.001 absorbance unit.
REFERENCES
1. Tietz, N.W., Fundamentals of Clinical Chemistry, W.B. Saunders Co., Philadelphia, 1986, p. 991.
2. Searcy, R.L., Reardon, J.E., and Foreman, J.A., Am. J. Med. Tech. 33, 15 (1967).
3. Talke, H., and Schuber, G.E., Klin. Wschr. 43:174 (1965)
4. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. Vol. 21, p.371D
(1975).
