Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

Mix 35 ml of solution A with 35 ml of solution B, and make up to 500 ml with
distilled water. Store in a brown bottle at room temperature.
3.Stock urea standard: Prepare 100 mg/dl urea solution in distilled water.
4.Working standard: Prepare 2.5 mg/dl working solution.

18.6 Procedure

1. Take a set of nine test tubes and label as blank, standard (S 1 – S 6 ), test for serum
   sample (Ts), and test for urine sample (Tu).


2. Add 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of working urea standard into the tubes
   labeled as S 1 to S 6 , respectively, and make thefinal volume 2 ml with distilled
   water.


3. In Ts add 0.2 ml diluted serum sample (1:10) and 0.4 ml of diluted urine sample
   (1:100) to the tube marked as TU, and make the volume of each tube to 2 ml with
   distilled water.


4. Add 2 ml of distilled water in blank tube, and then add 2 ml each of acid reagent
   and color reagent to all tubes.


5. Mix well and incubate all the tubes in boiling water bath for 20C and cool.


6. Measure the absorbance at 520 nm in a spectrophotometer.

Reagents Blank S 1 S 2 S 3 S 4 S 5 S 6 TS TU

Working urea

(ml)

• 0.1 0.2 0.4 0.6 0.8 1.0 ––

Amount of urea

(mg)

• 0.0025 0.005 0.010 0.015 0.020 0.025

Serum/urine

(ml)

–––––––0.2 0.4

Distilled water

(ml)

2 1.9 1.8 1.6 1.4 1.2 1 1.8 1.6

Acid reagent

(ml)

22 2222222

Color reagent

(ml)

22 2222222

Mix well and keep in boiling water bath for 20 min, and cool and read absorbance at 520 nm

16.6 Calculations

Plot a graph between amount of urea at x-axis and absorbance at y-axis. Then
extrapolate amount of urea from the given serum/urine sample from the graph
(include dilution factor for calculation). The urea concentration can also be calcu-
lated by using the following equation:

16.6 Calculations 69