How to Make a Blood Urea Nitrogen Test

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Three methods of how to make a blood urea nitrogen (BUN) test are shown below: Diacetyl Monoxime, Urograph and Modified Berthelot Enzymatic methods:

1. DIACETYL MONOXIME Method
   • This method utilizes the Fearon Reaction, wherein urea in hot solution of diacetyl monoxime condenses to form diazine derivative.

     Reagents to use

     • BUN Std (usually 15 mg/dl);
     • Serum or Plasma (Patient's Sample);
     • Distilled Water;
     • Acid Reagent (Diacetyl monoxime);
     • Color Reagent (Ferrithiocyanate).

     Equipment needed

     • Test tubes;
     • Pipettes;
     • Boiling water bath;
     • Iced cold water bath;
     • Cuvet;
     • Spectrophotometer.

     Procedure

     1. Prepare 3 to 10 mL test tubes
        • Blank - 0.05 mL Distilled water
        • Standard - 0.05 mL Std Solution
        • Test - 0.05 mL Serum or Plasma
     2. To all tubes add 3.0 mL of Acid Reagent and mix by lateral tapping;
     3. Then add Color reagent and mix well again;
     4. Place all tubes into an already boiling water bath for 15 minutes;
     5. Cool in iced water bath for 2 minutes;
     6. Let stand at room temperature for 1 minute;
     7. Read against Blank at 520 nm in Spectrophotometer

     Interpolation Formula

     (Optical Density of Test/Optical Density of Std)*(15 mg/dL as Concentration of Std)*(0.357)= BUN in mmol/L

     • 0.357 is the Conversion Factor for BUN to mmol/L

     Normal Value

     2.86 to 7.14 mmol/L (or 8 to 20 mg/dL)

2. UROGRAPH method
   • This method utilizes Physical and Chemical means via Paper Chromatography and Conway Microdiffusion respectively. Conway Microdiffusion involves the hydrolysis of Urea with buffered Urease, the liberation of ammonia gas and reaction with indicator system.

     Reagents to use

     • Urastrat strip;
     • Serum or Plasma.

     Equipment needed

     • Test tubes w/ Test Tube Rack;
     • Pipet;
     • Ruler or Caliper.

     Procedure

     1. Place 0.2 mL of serum or plasma at the bottom of a 5.0 mL test tube;
     2. Place Urastrat strip in the test tube with the lower portion touching the serum;
     3. Place the tube in a rack in straight position and let stand for 30 minutes;
     4. Read the blue colored band produced using the millimeter graduation of a ruler or a caliper.

     Compute as

     (Height of blue band in mm)*5 + 10 = (BUN in mg/dL)*(0.357)= BUN in mmol/L

     • 0.357 is the Conversion Factor for BUN to mmol/L

3. Modified Berthelot enzymatic method
   • This method utilizes the principle of urea hydrolysis. Urea is hydrolyzed in the presence of urease to carbon dioxide and ammonium ions.

     Reagents to use

     • Patient's serum;
     • Urea N standard (25 mg/dL);
     • Urea N-Zyme reagent (buffered urease);
     • Urea N Color reagent (sodium salicylates and sodium nitroferricyanide);
     • Urea N Base reagent (NaOH and sodium hypochlorite).

     Equipment needed

     • Test tubes w/ Test Tube Rack;
     • Pipettes;
     • Cuvet;
     • Spectrophotometer;
     • 37°C water bath.

     Procedure

     1. Prepare 3 to 10 mL test tubes labeled as Blank, Test and Standard;
     2. Place 0.5 mL urea N color reagent to all tubes and add sample as follows:
        • Blank - 0.5 mL distilled water
        • Standard - 0.5 mL std solution
        • Test - 0.5 mL serum or plasma
     3. Mix by gentle swirling and pre-warm at 37 °C water bath for 5 minutes;
     4. Add 2.0 mL urea N base reagent to all tubes, mix and return to 37 °C water bath for 5 minutes;
     5. Read at 630 nm against reagent blank in spectrophotometer.

     Compute as

     (Optical Density of Test/Optical Density of Std)*(15 mg/dL as Concentration of Std)=BUN in mg/dL*(0.357)= BUN in mmol/L

     • 0.357 is the conversion factor for BUN to mmol/L

     Normal value

     8 to 26 mg/dL

[edit] See also

• Wikipedia: w:Blood urea nitrogen