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

(Chris Devlin) #1

For Urine Dilute urine 1:10 with distilled water and prepare protein-freefiltrate the
same way as for serum.


24.5 Procedure



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

  2. Add 10, 20, 40, 60, 80, and 100μl of 20 mg % phosphorus standard in tubes
    labelled as S 1 to S 6 , respectively, and make thefinal volume 2.5 ml with 20%
    TCA. In blank add 2.5 ml of 20% TCA.

  3. Add 2.5 ml of protein-freefiltrate of serum and urine into the tubes labelled at TS
    and TU, respectively.

  4. Add 1 ml of ammonium molybdate reagent and 0.2 ml ANSA reagent to all the
    tubes and vortex. Keep at room temperature for 20 min and read absorbance at
    700 nm.


Reagents Blank S 1 S 2 S 3 S 4 S 5 S 6 TS TU
Phosphorus standard,
20 mg % (μl)


  • 10 20 40 60 80 100 ––


Phosphorus amount (μg) – 2 4 8 12 16 20 ––
Protein-freefiltrate of
serum/urine (ml)

–––––––2.5 2.5

20% TCA (ml) 2.5 2.49 2.48 2.46 2.44 2.42 2.40 ––
Ammonium molybdate
(ml)

1 11111111

ANSA (ml) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Mix well and keep for 20 min at room temperature, read absorbance at 700 nm

22.8 Calculations


Plot a graph by taking phosphorus amount at x-axis vs absorbance at y-axis, and
extrapolate its concentration from the given serum/urine sample from the graph.The
amount of phosphorus extrapolated from graph will be in 250μl of serum/urine
sample since 2.5 ml of protein-freefiltrate was used for analysis. The phosphorus
amount can also be calculated in serum and urine samples by using equation:


Phosphorus amount in serum
¼

ODof testamount of standardðÞμg 100
ODof standardvolume of sample 0ðÞ:25ml 1000

¼xmg=dl

22.8 Calculations 91

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