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

18.4 Reagents........................................

1.2/3NH 2 SO 4

2.10% sodium tungstate:Dissolve 10 g sodium tungstate infinal volume of
100 ml distilled water.
3.0.04 M picric acid:Dissolve 9.3 g pure dried picric acid in water, and makefinal
volume to 1000 ml.
4.0.75 N NaOH
5.Stock creatinine standard 100 mg/dl:Dissolve 100 mg of pure creatinine in
100 ml of 0.1 N HCl.
6.Working creatinine standard:Prepare 1 mg/dl solution in distilled water.

18.5 Preparation of Protein-Free Filtrate

For serum add 4 ml of 2/3 N H 2 SO 4 to 0.5 ml of serum, and add 0.5 ml of 10%
sodium tungstate solution. Mix well and keep for 10 min. Centrifuge at 3000 rpm for
5 min. Supernatant is used for the estimation of creatinine.
For urine: dilute urine 1:10 with distilled water. Preparefiltrate as with serum.

21.6 Procedure

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


2. Add 0.5 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, and 3 ml of working 1 mg/dl creatinine
   standard to the tubes labelled as S 1 to S 6 , respectively, and make the volume 3 ml
   with distilled water. To blank tube, add 3 ml of distilled water.


3. Add 3 ml of protein-freefiltrate of serum and urine samples in respective tubes
   followed by 1 ml each of NaOH and picric acid.

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

Creatinine working standard (ml) – 0.5 1 1.5 2 2.5 3 ––

Creatinine amount (μg) – 5 1015202530––

Protein-freefiltrate of serum/urine

(ml)

––––––– 33

Distilled water (ml) 3 2.5 2 1.5 1 0.5 –––

0.75 N NaOH (ml) 1 1 1 1 1 1 1 1 1

Picric acid (ml) 1 1 1 1 1 1 1 1 1

Mix well and keep for 10 min at room temperature; read absorbance at 520 nm

18.6 Procedure 77