UREA

(Modified Berthelot Method)

For the determination of Urea in serum and plasma
(For In vitro Diagnostic use only)

SUMMARY:

Urea is the end product of the protein metabolism. It is Synthesized in the liver from ammonia produced by
the metabolism of amino acids. It is transported by the blood to the kidneys from where it is excreted.

PRINCPLE:
Urease
Urea + H2O Ammonia + CO2

Ammonia + Phenolic Green Colored

Chromogen + Hypochlorite Complex
The intensity of the color formed is directly proportional to the amount of Urea present in the sample.

CONTENTS:

Pack Size Buffer Reagent(A1) Enzyme Reagent(A2) Chromogen Reagent(A3) Standard (S)

2x60ml 1x60ml 1x3ml 1x60ml 5ml

2x100ml 1x100ml 1x5ml 1x100ml 5ml

STORAGE/STABILITY:

Reagent is stable at 2 – 8˚C till the expiry mentioned on the label.

Urea Standard is stable at 2-8˚C till the expiry mentioned on the label.

REAGENT PREPARATION:

Reagents are ready to use for the following procedure.

WORKING REAGENT PREPARATION(WR):

For the flexibility and convenience in performing large assay series , a working enzyme reagent may be made by
pouring one bottle of A2 (Enzyme reagent) into one bottle of A1 (Buffer reagent).Working Reagent is stable for 16
weeks at 2 – 8˚C

SAMPLE:

Serum or Plasma. Urea are reported to be stable in the sample for 5 days when stored at 2-8˚C.
General System Parameter:

Reaction Mode End Point Sample Volume 10 ul

Wavelength 570 nm Reagent volume 1000ul(WR)+1000ul(A3)
Blank Reagent Standard 40 mg/dl
Incubation 5 min 37˚C/10 min R.T Reaction Slope Increasing
Delay Time 5 Sec Linearity 300 mg/dl
Read Time 5 Sec Units mg/dl

ASSAY PROCEDURE:
Wavelength/ Filter : 570 nm (Hg 578 nm)/Yellow
Temperature : R.T/37˚C
Light Path : 1 cm
Pipette into clean dry test tube labeled as Blank(B),Standard (S) and Test(T)

Addition Sequence B(ul) S(ul) T(ul)

Working Reagent(WR) 1000 1000 1000
Standard(S) - 10 -
Sample - - 10
Mix and incubate for 3 min at 37˚C / 5 min at R.T

Chromogen Reagent 1000 1000 1000

Mix well and incubate at 37˚C for 5 min or at R.T. for 10 min. Measure the absorbance of the standard
(Absorbance of Standard) and test Sample(Absorbance of Test) against reagent blank at 578 nm
(570 – 620 nm) within 10 hours at R.T

CALCULATION:
Absorbance of Test
Urea in mg/dl =--------------------------- × 40
Absorbance of Standard

NORMAL REFERENCE VALUES:

Serum and Plasma : 14 - 40 mg/dl
urine : 20 g/l
It is recommended that each laboratory establish its own normal range representing its patient population.

LINEARITY:
This procedure is linear up to 300 mg/dl. If values exceed this limit , dilute the sample with normal saline(NaCl
0.9%) and repeat assay. Calculate the value using the proper dilution factor.

NOTE:
1. Enzyme Reagents (A2) may appear slightly hazy, but after mixing it with Buffer Reagent (A1) its haziness
disappears and this does not affect the performance of kit..
2. Any contamination by ammonia or ammonium salts lead to erroneous results. Hence plasma should not be
collected within fluoride or Heparin ammonium salts.
3. The working enzyme reagent is not stable at elevated temperatures and should be stored back at 2-8 O C
immediately after use.
4. The chromogen reagent contains chlorine. The bottles should be opened only when required and closed tightly
after use to prevent the loss of active chlorine.

REFERENCES:
1.Berthelot,M.P.E,(1856)Report chim.Appl.2884
2.Fawcett,j.k.Scott,J.E.,(1960) J.Chim. Pathol .13:156