Urea ( Berthelot Method )

 Clinical significance :-

                        Urea is the end product of the protein metabolism. It is synthesized in the liver form  the ammonia produced by the catabolism of amino acids. It is transported by the blood to the kidney from where it is excreted. Increased level are found in renal diseases, urinary obstructions,  shock, congestive heart failure and burns. Decrease level are found in liver failure and due to  pregnancy.

Principle :-

            Urea hydrolyzes urea to ammonia and CO2 . The ammonia formed  react with a  phenolic chromogen and hypochlorite to form a green coloured complex . Intensity of the colour formed  is directly proportional to the amount of Urea present in the sample.

 Material required :-

•  Clean and dry glassware.                                     
• Laboratory glass Pipettes or  micro Pipettes and tips. 
• Colorimeter.

 Normal Value :- 

                    10 - 50  mg/dl  

                        

Sample :-

           Serum .

Procedure :-

               Pipette into clean and dry test tubes labeled as Blank ( B ) , Standard ( S ), and Test ( T ) :

Addition sequence	B	S	T
Reagent	1 ml	1ml	1 ml
Standard	-	10 ul	-
Sample	-	-	10 ul

Mix well and incubate at 37 ‘ C for 5 min.

Chromogebn Reagent

1 ml

1ml

1ml

 

.

 Mix well  and  incubate  at  37 ‘ C for 05  mins. Measure absorbance of the standard ( Abs . S ) and Test ( Abs . T ) against Reagent blank at 505 nm.

 

   Calculation  :-

                                              Abs . T

                    Uric Acid ( mg/dl ) =    ..............  * 40

                                              Abs . S   

Preparation and reagent and stability :-

                                        All Reagent are ready to use and are stable till the expiry date mentioned on the label , when stored at 2' to 8' C.

Quality Control :-

                  For accuracy it is necessary to run known controls with every assay.

Limitation & Precautions :-

 

1. Storage conditions as mentioned on the kit to be adhered.
2. Do not freeze or expose the reagent to high temperature and protect from direct as it may affect the performance of the kit.
3. Use clean glassware free from dust .
4. Before the assay bring all reagents to room temperature.

 General System Parameters:-

          Reaction type           : End point 

          Wave length             : 600 nm ( 600 - 630 nm )

          Temperature             : 37'C

          Incubation              : 5 + 5 minutes 

          Reagent Volume          : 1.0 ml

          Sample Volume           : 10 ul 

          Standard concentration  : 40 mg/dl

          Zero setting            : Reagent Blank

          Light Path              : 1.0 cm