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

27.5 Procedure

1. Take a set of nine test tubes, and label them as blank, standard (S 1 – S 6 ), test (T),
   and control (C).


2. Add 10, 20, 40, 60, 80, and 100μl of pyruvic acid standard to the test tubes
   marked as S 1 to S 6 , respectively.


3. Make volume in each tube to 100μl using phosphate buffer.


4. Add 0.5 ml of buffered substrate to all the tubes. Then mix and incubate for 3 min
   at 37C.


5. Add 100μl of serum to test tube labelled as T. Mix and note time.


6. Incubate the tubes at 37C for 30 min for ALT and 1 h for AST. Keep at room
   temperature for 20 min.


7. Immediately after adding DNPH, add 100μl of serum in control tube.


8. Add 5 ml of 0.4 N NaOH to all tubes, and mix and keep at room temperature for
   10 min.


9. Read absorbance at 540 nm against blank.

Note For AST estimation the process is the same except the following:

1. Use AST substrate


2. Incubate for 60 min

Reagents Blank S 1 S 2 S 3 S 4 S 5 S 6 TC

Pyruvic acid standard (μl) – 10 20 40 60 80 100 ––

Pyruvate amount (mmol) – 0.1 0.2 0.4 0.6 0.8 1 ––

Phosphate buffer (μl) 100 90 80 60 40 20 –––

Buffered substrate for ALT/AST

(ml)

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Mix and incubate at 37C for 3 min

Serum (ml) –––––––0.1 –

Mix well and incubate at 37C for 30 min in case of ALT and for 1 h in case of AST

DNPH (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Serum (ml) ––––––––0.1

Mix well and incubate at 37C for 20 min

NaOH (ml) 5 555555 55

Mix well and incubate at 37C for 10 min, then read absorbance at 540 nm

25.5 Procedure 105