Urea liquiUV Pipetting Scheme

Please use only the standard recommended by HUMAN (enclosed in the

GLDH Method kit or separately available with [REF] 10104).
Fully Enzymatic Method for Kinetic Determi- Reagent Start Procedure
nation of Urea Pipette into cuvettes Reagent blank (RB) Sample or [STD]
Package Sizes Sample / [STD] --- 10 µl
[REF] 10521 8 x 50 ml Complete Test Kit [ENZ] 1000 µl 1000 µl
10104 9 x 3 ml Standard Mix, incubate for approx. 1 minute.
[IVD] [SUB] 250 µl 250 µl

Method 1, 2, 3 Mix, read absorbance of sample/[STD]after 30 seconds (A1), start timer

simultaneously and read again after exactly 1 minute (A2). Calculate
Urea is hydrolysed in the presence of water and urease to produce am-
the absorbance difference:
monia and carbon dioxide. The ammonia from this reaction combines
with 2-oxoglutarate and NADH in the presence of glutamate-dehydroge- ASample/[STD] = (A2 A1) - ARb.
nase (GLDH) to yield glutamate and NAD+. The test has been optimised so
that the GLDH is the rate limiting enzyme. The decrease in absorbance is Sample Start Procedure
proportional to the urea concentration within the given time intervals. As Pipette into cuvettes Reagent blank (RB) Sample or [STD]
the kinetic is very fast this test is preferably designed for analyzer Sample / [STD] --- 10 µl
application.
Working reagent 1000 µl 1000 µl
Reaction Principle Mix, read absorbance of sample/standard after 30 seconds (A1), start
Urease timer simultaneously and read again after exactly 1 minute (A2). Cal-
Urea + 2 H2O 2 NH4+ + CO32- culate the absorbance difference:
GLDH ASample/[STD] = (A2 - A1) - ARb.
2-oxoglutarate + NH4+ L-glutamate + H2O + NAD+
Calculation of the Urea Concentration
+ NADH
Serum, Plasma
Contents C = 80 x A Sample / A [STD] [mg/dl] or
[ENZ] 8 x 40 ml Enzymes C = 13.3 x A Sample / A [STD] [mmol/l]
Tris buffer (pH 7.8) 125 mmol/l Urine
ADP 0.88 mmol/l
C = 80.8 x A Sample / A [STD] [mg/dl] or
Urease 20 kU/l
C = 1340 x A Sample / A [STD] [mmol/l]
GLDH 0.3 kU/l
Sodium Azide 0.095 % Conversion Factors for BUN / Urea
[SUB] 8 x 10 ml Substrate C (BUN) = 0.466 x C (urea) C (urea) = 2.14 x C (BUN)
2-oxoglutarate 25 mmol/l
NADH 1.25 mmol/l Performance Characteristics
Sodium Azide 0.095 % Linearity
[STD] 1 x 3 ml Standard The test is linear up to 300 mg/dl or 50 mmol/l.
Urea 80 mg/dl The linearity depends on the respective analyzer application.
or 13.3 mmol/l Dilute samples with higher concentrations 1 + 1 with distilled water and
Sodium Azide 0.095 % repeat the assay. Multiply the result by 2.
Typical performance date can be found in the Verification Report, acces-
Reagent Preparation sible via
The reagents are ready for use and can directly be applied on automated
analyzers (Reagent start procedure). www.human.de/data/gb/vr/su-urluv.pdf or

For sample start procedure working reagent is prepared by mixing 4 parts www.human-de.com/data/gb/vr/su-urluv.pdf
of [ENZ] with 1 part of [SUB], e.g. 40 ml [ENZ] + 10 ml [SUB].
Quality Control
Reagent Stability All control sera with urea values determined by this method can be
The individual reagents are stable, even after opening, up to the stated employed.
expiry date when stored at 2...8°C. Contamination of the reagents must We recommend to use our control sera HumaTrol based on animal serum
be strictly avoided. or our SERODOS based on human serum.
The working reagent is stable for 5 days at 15...25°C and for 4 weeks at
Automation
2...8°C.
Proposals to apply the reagents on analyzers are available on request.
Specimen Each laboratory has to validate the application in its own responsibility.
Serum, plasma, except ammonium heparinate plasma, or urine.
Notes
Dilute urine 1+100 with dist. water (results x 101). All reagents contain sodium azide (0.095%) as preservative. Do not swal-
low. Avoid contact with skin and mucous membranes.
Assay
Wavelength: 340 nm, Hg 334 nm, 365 nm References
Optical path: 1 cm 1. Kassirer J. P., New Eng. J. Med. 285, 385 (1971)
Temperature: 25°C, 30°C or 37°C 2. Talke H., Schubert G. E., Klin. Wochenschr. 43, 174 (1965)
Measurement: Against reagent blank (RB). Only one reagent blank 3. Tietz N. W., Fundamentals of Clinical Chemistry, 3rd. Edition (1987),
per series is required. 676 - 679, W. B. Saunders Company Philadelphia
2-point kinetic 4. MacKay E. M., MacKay L. L, J. Clin. Invest. 4, 295 (1927)
4, 5 5. Sarre H., Nierenkrankheiten, Georg Thieme Verlag Stuttgart (1959)
Reference Values
Serum: 10 - 50 mg/dl
Urine: 20 - 35 g/24h
or
or
1.7 - 8.3 mmol/l
333 - 583 mmol/24h
SU-URLUV INF 1052101 GB 02-2011-09 |

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