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    174 - 13 UREA PDF - 28-Euro Procedure Sheet

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    P Vijaya
    1) The document provides instructions for using a urea/BUN reagent kit to measure urea levels in serum, plasma or urine samples. Urea is hydrolyzed by urease and converted to ammonia, which is then converted to glutamate by glutamate dehydrogenase in the presence of NADH, allowing the reaction to be monitored kinetically. 2) Interfering substances include lipidemia, bilirubin, hemoglobin and some drugs. Fluorides used as anticoagulants can inhibit the urease. 3) The reagent contains buffered urease, glutamate dehydrogenase, cofactors and a urea standard. The procedure involves incubating the

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    174 - 13 UREA PDF - 28-Euro Procedure Sheet

    Uploaded by

    P Vijaya
    105 views2 pages
    1) The document provides instructions for using a urea/BUN reagent kit to measure urea levels in serum, plasma or urine samples. Urea is hydrolyzed by urease and converted to ammonia, which is then converted to glutamate by glutamate dehydrogenase in the presence of NADH, allowing the reaction to be monitored kinetically. 2) Interfering substances include lipidemia, bilirubin, hemoglobin and some drugs. Fluorides used as anticoagulants can inhibit the urease. 3) The reagent contains buffered urease, glutamate dehydrogenase, cofactors and a urea standard. The procedure involves incubating the

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    174_13 UREA PDF_28-Euro Procedure Sheet

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    1) The document provides instructions for using a urea/BUN reagent kit to measure urea levels in serum, plasma or urine samples. Urea is hydrolyzed by urease and converted to ammonia, which is then converted to glutamate by glutamate dehydrogenase in the presence of NADH, allowing the reaction to be monitored kinetically. 2) Interfering substances include lipidemia, bilirubin, hemoglobin and some drugs. Fluorides used as anticoagulants can inhibit the urease. 3) The reagent contains buffered urease, glutamate dehydrogenase, cofactors and a urea standard. The procedure involves incubating the

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    105 views2 pages

    174 - 13 UREA PDF - 28-Euro Procedure Sheet

    Uploaded by

    P Vijaya
    1) The document provides instructions for using a urea/BUN reagent kit to measure urea levels in serum, plasma or urine samples. Urea is hydrolyzed by urease and converted to ammonia, which is then converted to glutamate by glutamate dehydrogenase in the presence of NADH, allowing the reaction to be monitored kinetically. 2) Interfering substances include lipidemia, bilirubin, hemoglobin and some drugs. Fluorides used as anticoagulants can inhibit the urease. 3) The reagent contains buffered urease, glutamate dehydrogenase, cofactors and a urea standard. The procedure involves incubating the

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    UREA/BUN BR

    REF 1158005
    2 x 50 mL

    REF 1158010
    3 x 100 mL

    CONTENTS
    R1. Reagent 2 x 40 mL
    R2. Reagent 1 x 20 mL
    CAL. Standard 1 x 3 mL

    CONTENTS
    R1. Reagent 3 x 80 mL
    R2. Reagent 1 x 60 mL
    CAL. Standard 1 x 3 mL

    UREA/BUN BR
    Urease/GlDH
    UV enzymatic method
    KINETIC

    For in vitro diagnostic use only


    PRINCIPLE

    INTERFERENCES

    Urea is hydrolyzed by urease to ammonia and carbon dioxide. The


    ammonia is converted to glutamate by glutamate dehydrogenase
    1,2
    (GlDH) in the presence of NADH and oxoglutarate.
    The reaction is monitored kinetically at 340 nm by the rate of
    decrease in absrbanse resulting from the oxidation of NADH to
    +
    NAD , proportional to the concentration of urea present in the
    sample.

    Lipemia (intralipid < 5 g/L) does not interfere.


    Bilirubin (40 mg/dL), hemoglobin (< 4 g/L), do not interfere.
    3
    Other drugs and substances may interfere .
    Contamination of glassware and water by ammonia, will give
    falsely elevated results.
    Fluorides used commonly as anticoagulants inhibit the urease
    4
    of the substrate .

    UREASE
    2 NH4+ + CO2

    Urea + H2O
    GlDH
    2-Oxoglutarate + NH4+ + 2 NADH

    Glutamate + 2 NAD+ + 2 H2O

    REAGENT COMPOSITION
    R1

    R2
    CAL

    MATERIALS REQUIRED
    Photometer or spectrophotometer with a thermostatted cell
    compartment set at 37C, capable of reading at 340 nm.
    Stopwatch, strip-chart recorder or printer.
    Cuvettes with 1-cm pathlength.
    Pipettes to measure reagent and samples.

    Buffered Urease/GlDH. TRIS buffer 125 mmol/L pH


    7.4, 2-oxoglutarate 10 mmol/L, urease > 140 U/mL,
    glutamate dehydrogenase > 120 U/mL, Biocides.
    Coenzyme. NADH 1.50 mmol/L.
    Urea standard. Urea 50 mg/dL (8.3 mmol/L).
    Organic matrix based primary standard. Concentration
    value is traceable to Standard Reference Material 909b.

    PROCEDURE
    1. Preincubate working reagent, samples and standard to reaction
    temperature.
    2. Set the photometer to 0 absorbance with distilled water.
    3. Pipette into a cuvette:
    Reaction temperature

    STORAGE AND STABILITY


    Store at 2-8C.
    All the kit compounds are stable until the expiry date stated on the
    label. Do not use reagents over the expiration date.
    Store the vials tightly closed, protected from light and prevented
    contaminations during the use.
    Discard If appear signs of deterioration:
    - Presence of particles and turbidity.
    - Blank absorbance (A) at 340 nm < 1.100 in 1cm cuvette.

    37C

    Working reagent

    1.0 mL

    Sample or standard

    10 L

    4. Mix gently by inversion. Insert cuvette into the cell holder and
    start stopwatch.
    5. Record the absorbance at 340 nm exactly after 30 seconds (A1)
    and exactly 90 seconds later (A2).
    6. Calculate the difference between absorbances.

    CALCULATIONS

    REAGENT PREPARATION
    Serum, plasma
    Working reagent. Mix 4 mL of R1 + 1 mL of R2. Stable for 2 months
    at 2-8C.

    (A1 - A2 ) Sample
    x C Standard = mg/dL urea

    SAMPLES
    Serum or heparinized plasma free of hemolysis and urine (see
    Notes). Other anticoagulants (ammonium heparinate or double
    oxalate of potassium and ammonium ) must not be used.
    Urea in serum, plasma or urine is stable 7 days at 2-8C.
    Freeze for longer storage.

    QUALITY SYSTEM CERTIFIED

    ISO 9001 ISO 13485

    (A1 - A2 ) Standard
    Samples with concentrations higher than 500 mg/dL should be
    diluted 1:2 with saline and assayed again. Multiply the results by 2.
    Urine
    Dilute the sample 1:50 with distilled water and multiply the result by
    50.

    LINEAR CHEMICALS S.L. Joaquim Costa 18 2 planta. 08390 Montgat, Barcelona, SPAIN
    Telf. (+34) 934 694 990 Fax. (+34) 934 693 435. website www.linear.es

    NOTES

    If results are to be expressed as SI units apply:


    mg/dL x 0.1665 = mmol/L
    To convert urea mass units to those of urea nitrogen apply:
    mg/dL x 0.467 = mg/dL BUN

    REFERENCE VALUES 5

    Collect a 24-hour urine specimen into a plastic bottle free of


    preservatives. Keep the sample refrigerated to minimize urea
    hydrolysis by microorganisms or other agents.

    This method may be used with different instruments. Any


    application to an instrument should be validated to demonstrate
    that results meet the performance characteristics of the
    method. It is recommended to validate periodically the
    instrument. Contact to the distributor for any question on the
    application method.

    Clinical diagnosis should not be made on findings of a single


    test result, but should integrate both clinical and laboratory
    data.

    Serum, plasma
    Newborns (< 10 days)

    6.4 - 53.5 mg/dL (1.1 - 9.0 mmol/L)

    Adults (12-60 years)

    15 - 40 mg/dL (2.5 - 6.6 mmol/L)

    In adults over 60 years of age, the reference interval is 17-50 mg/dL


    (2.8-8.3 mmol/L) and the concentrations tend to be slightly higher in
    males than in females.

    ANALYTICAL PERFORMANCE

    Urine
    Adults (normal diet)

    26 - 43 g/24-h (428 - 714 mmol/24-h)

    - Detection Limit : 1.14 mg/dL


    - Linearity : Up to 500 mg/dL

    A high-protein diet causes significant increases in plasma urea


    concentrations and urinary excretion.
    It is recommended that each laboratory establishes its own
    reference range.

    QUALITY CONTROL
    The use of a standard to calculate results allows to obtain an
    accuracy independent of the system or instrument used.
    To ensure adequate quality control (QC), each run should include a
    set of controls (normal and abnormal) with assayed values handled
    as unknowns.
    REF

    REF

    1980005 HUMAN MULTISERA NORMAL


    Borderline level of urea. Assayed.
    1985005 HUMAN MULTISERA ABNORMAL
    Elevated level of urea. Assayed.

    If the values are found outside of the defined range, check the
    instrument, reagents and procedure.
    Each laboratory should establish its own Quality Control scheme
    and corrective actions if controls do not meet the acceptable
    tolerances.

    CLINICAL SIGNIFICANCE
    Urea is the chief end product of protein metabolism in the body.
    The importance of the urea concentration in blood lies in its value
    as an indicator of kidney function.
    Azotemia ( an abnormal increase in plasma urea level) is seen
    mainly in renal disorders, dehydration, increase protein catabolism,
    high-protein diets, or gastrointestinal hemorrhage. There are two
    types of azotemia. The first, prerenal azotemia, is caused by
    impaired perfusion of the Kidneys due to decreased cardiac output
    or for any of the former causes. The second, postrenal azotemia, is
    caused by an obstruction in the urine outflow such as
    nephrolithiasis, prostatism, and tumors of the genitourinary tract.
    The clinical significance of the urea level in plasma is usually
    determined in conjugation with the plasma creatinine level. In
    prerenal azotemia, an increase in the plasma urea level is usually
    associated with a normal plasma creatinine level, where as in
    postrenal azotemia, there is an increase in both the urea and the
    plasma creatinine levels. A decrease in the urea plasma level may
    be associated with acute dehydration, malnutrition, and pregnancy.

    - Precision:
    mg/dL

    Within-run

    Between-run

    Mean

    47.8

    121.6

    47.8

    121.6

    SD

    0.43

    0.87

    0.82

    2.25

    CV%

    0.91

    0.71

    1.72

    1.85

    10

    10

    10

    10

    - Sensitivity : 3 mA/min / mg/dL Urea.


    - Correlation. This assay (y) was compared with a similar commercial
    method (x). The results were:
    N =57
    r = 1.00
    y = 1.03x + 0.86
    The analytical performances have been generated using on
    automatic instrument. Results may vary depending on the
    instrument.

    REFERENCES
    1. Talke, H., and Schubert GE. Klin. Wochenschr. 43 : 174
    (1965).
    2. Gutman, I., and Bergmeyer, H.U. Methods of Enzymatic
    Analysis, Ed. H.U. Bergmeyer, Verlag Chemie, A.P. 2 ed. 4 :
    1794 (1974).
    3. Young DS. Effects of drugs on clinical laboratory tests, 5th ed.
    AACC Press, 2000.
    4. Patton, C.S., and Crouch, S.R. Anal. Chem. 49 : 464 (1977).
    rd
    5. Tietz. N.W. Clinical Guide to Laboratory Tests, 3 Edition.
    W.B. Saunders Co. Philadelphia, PA. (1995).

    B1158-2/0901
    R1.ing

    QUALITY SYSTEM CERTIFIED

    ISO 9001 ISO 13485

    LINEAR CHEMICALS S.L. Joaquim Costa 18 2 planta. 08390 Montgat, Barcelona, SPAIN
    Telf. (+34) 934 694 990 Fax. (+34) 934 693 435. website www.linear.es

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