UREA
UV Method (5 + 1 Reagent)
METHOD and PRINCIPLE
Enzymatic 2-Point determination in the UV-range,
according to the following reactions:
Urease
Urea + H20
2NH3 + CO2
GLDH
2NH4+ + 2-Ketoglutarate + 2NADH
2L-Glutamate +
2NAD+ + 2H20
REFERENCE VALUES
In Serum / Plasma
Adults
Global
Women
Men
Children
1 - 13 Years
14 - 19 Years
(GLDH = Glutamate dehydrogenase
Urea/Creatinine-Ratio
in Serum
REAGENTS
Composition (concentrations in the test)
25 – 40 [(mmol/l)/(mmol/l)]
R1:
R2:
Standard :
TRIS-Buffer( pH 7,8)
2-Oxoglutarate
ADP
Urease
GLDH
(Glutamatdehydrogenase)
NADH
120 mmol/l
7 mmol/l
0,6 mmol/l
 6 kU/l
 1 kU/l
0,25 mmol/l
50 mg/dl (8,33 mmol/l)
PRECAUTIONS
-
For in vitro diagnostic use only.
The reagents contain < 0,95 g/L sodium azide.
Avoid contact with skin and/or mucous membranes
-
STABILITY OF REAGENTS
When stored at 2-8° C and protected from light, the
reagents are stable until the expiry date stated on
the label
PREPARATION AND STABILITY
OF WORKING REAGENT
• One-reagent procedure
Mix 5 volumes of R1 with 1 volume of R2.
(Let monoreagent stand for 10 min before use)
Stability :
[mg/dl]
[mmol/l]
17- 43
15 - 43
18 - 55
2,8 - 7,2
2,6 - 6,7
3,0 - 9,2
11 - 36
18 - 45
1,8 - 6,0
2,9 - 7,5
= 20 – 35 [(mg/dl)/(mg/dl)]
26 – 43 g/day (0,43 – 0,72 mol/day)
In Urine
Note: It is recommended for each laboratory to establish and
maintain its own reference values. The given data here are only
an indication.
ASSAY PROCEDURES
This reagent can be used manually (see method
below) and on most analyzers. Applications are
available on request.
Wavelength : 340 nm, Hg 334nm, Hg 365 nm
Temperature
:
37°C
Cuvette
:
1 cm light path
Read against distilled water
• One-reagent procedure
Working reagent
Sample/Standard
1 mL
10µL
Mix and read the change of absorption (A) between
30 seconds and 90 seconds:
A = A 1 - A 2
• Two-reagent procedure
5 days at 15-25°C
4 weeks at 2-8°C
Reagent R1
1000 µL
Sample/Standard
10 µL
Mix and incubate about 5 min , the add :
Reagent R2
200 µL
SAMPLES
Serum, Plasma (EDTA / heparin)
Urine diluted 1+100 with distilled water.
(Do not use anticoagulants containing fluoride or ammonium
ions).
Mix and read the change of absorption (A) between
30 seconds and 90 seconds:
A = A 1 - A 2
CALCULATION
A Sample
A Standard
x C
mg/dL
g/L
mmol/L
C =
50
C =
0.5
C = 8.325
(C = standard concentration)
For Urine note dilution, i.e. multiply by dilution factor
Page 1/2
Greiner Diagnostic GmbH - Unter Gereuth 10 – D-79353 Bahlingen – Germany
www.greiner-diagnostic.com
BIBLIOGRAPHY
CALIBRATORS & CONTROLS
For the calibration of automated analyzers Greiner
Multicalibrator is recommended, for quality control
use Greiner normal and abnormal control, Unitrol I
and Unitrol II.
1.
2.
3.
PERFORMANCE DATA
- Analytical range
The reagent is linear from 2 – 400 mg/dL (0.33 –
66.6 mmol/L) in Serum / Plasma or 30 g/dL (5.0
mol/L) in urine.
Thomas L. Clinical Laboratory Diagnostics. 1st ed.
Frankfurt: TH-Books Verlagsgesellschaft; 1998,
p. 374-7.
Burtis CA, Ashwood ER, editors. Tietz Textbook of
Clinical Chemistry. 3rd ed. Philadelphia:
W.B Saunders Company; 1999. p. 1838.
Talke H, Schubert GE. Enzymatische Harnstoffbestimmung in Blut und Serum im optischen Test
nach Warburg. Klin Wschr 1965;43:174-5.
SYMBOLS USED
- Detection limit
The detection limit is equal to 2 mg/dL
For in vitro diagnostic medical use
- Precision
Within-run reproducibility
N = 11
Mean
mg/dL
Control 1
42,4
SD
mg/dL
0,76
CV
%
1,79
Batch Code
Use by
Control 2
145
1,22
0,84
Patient
86,1
2,39
2,77
Between-run reproducibility
N = 11
Mean
mg/dL
Control 1
42,4
Temperature limitation
SD
mg/dL
0,55
CV
%
1,29
Control 2
144
1,50
1,05
Patient
86,5
1,44
1,66
- Correlation
A comparative study has been performed between the
Greiner method and another commercial reagent on 26
human serum samples. The parameters of linear
regression are as follows:
y = 1.000x + 1.450 mg/dl
r = 0.999
INTERFERENCES
-
Ascorbic acid: no interference until 20 mg/dL
-
Bilirubin: no interference until 40 mg/dL
-
Hemoglobin: no interference until 500 mg/dL
-
Triglycerides: no interference up to 2000 mg/dL
Page 2/2
Greiner Diagnostic GmbH - Unter Gereuth 10 – D-79353 Bahlingen – Germany
www.greiner-diagnostic.com