20.3 Principle........................................
Proteins are precipitated with tungstic acid and are removed by centrifugation.
Protein-freefiltrate of serum or urine containing uric acid is oxidized to allantoin
by phosphotungstic acid and is reduced to tungsten blue in alkaline medium. The
blue color thus produced is measured at 700 nm and compared with that of uric acid
standard.
Uric acidþPhosphotungstic acidþO 2 þ2H 2 O!AllantoinþCO 2
Allantoin!alkali
reduction
Tungsten blue20.4 Reagents........................................
1.10% sodium tungstate aqueous solution.
2.3/2NH 2 SO 4
3.Phosphotungstic acid: Dissolve 10 g of sodium tungstate in about 150 ml of
distilled water. Add 8 ml of 85% H 3 PO 4 and put some glass beads. Reflux for
2 – 3 h. Cool to room temperature and then add 50 ml of distilled water containing
8 g of dissolved lithium sulfate. Mix and makefinal volume up to 250 ml with
distilled water and store in colored bottle.
4.7% aqueous solution of Na 2 CO 3.
5.Uric acid standard (20 mg/dl): Dissolve 12 mg of lithium carbonate in warm
water and add 20 mg of uric acid and dissolve by heating. Make volume up to
100 ml with distilled water.
22.7 Procedure
Protein-freefiltrate of serum is prepared by adding 0.5 ml serum in a test tube
containing 4 ml of 3/2 N H 2 SO 4 and added with 0.5 ml of 10% sodium tungstate.
Mix well and keep for 5 min and centrifuge at 3000 rpm for 5 min.
Protein-freefiltrate of urine is prepared by diluting urine 1:10 with distilled water
and following same process as that of serum. Add uric acid standard in tubes labelled
as (S 1 – S 6 ) and mix other reagents according to protocol given in table.
Reagents Blank S1 S2 S3 S4 S5 S6 TS TU
Working uric acid
standard (μl)- 50 100 150 200 250 500 ––
Uric acid amount (μg) -- 10 20 30 40 50 100 ––
Protein-freefiltrate of
serum/urine (ml)––––––– 33Distilled water (ml) 3 2.95 2.90 2.85 2.80 2.75 2.5 ––
(continued)82 20 To Determine the Uric Acid Concentration in Serum and Urine