22.2 Specimen Requirements
Fasting serum sample is preferred. Avoid hemolysis since blood cell contains high
amount of phosphate (RBCs contain about 40 mg/dl of phosphorus). Sample is
stable for several days at 4C. For urine collect 24 h samples in acid-washed
detergent-free container, and acidify with HCl after collection. Stable for 6 months.
22.3 Methodology....................................
Phosphorus content is measured by following the method of Fiske and Subbarow.
22.4 Principle........................................
Proteins are precipitated with TCA and are removed by centrifugation. Protein-free
filtrate contains phosphorus that reacts with molybdic acid to form phosphomolybdic
acid. The hexavalent molybdenum of the phosphomolybdic acid is reduced by 1,2,4
amino-naphthol-sulfonic acid (ANSA). The color thus produced is directly propor-
tional to the amount of phosphorous present in the sample.
22.5 Reagents........................................
1.20% TCA: Dissolve 20 g of TCA in 100 ml distilled water.
2.Ammonium molybdate reagent
Sol. A: 1 N HCl–11.5 ml conc. HCl is diluted to 100 ml with distilled water.
Sol. B: Ammonium molybdate solution–Dissolve 6.25 g of ammonium molyb-
date in water, and make volume to 150 ml with distilled water.
Prepare ammonium molybdate reagent by mixing Sol. A and B with constant
mixing.
3.ANSA reagent: Dissolve 250 mg of ANSA powder in a solution containing
97.5 ml of 15% aqueous solution of sodium bisulfite and 2.5 ml of 20% sodium
sulfite.
4.Phosphorus standard (20 mg %):Dissolve 11.48 mg of Na 2 HPO 4 .2H 2 Oin
distilled water to makefinal volume 100ml.
22.6 Preparation of Protein-Free Filtrates
For SerumMix 0.5 ml serum with 4.5 ml 20% TCA and allow standing for 10 min
at room temperature. Then centrifuge at 3000 rpm for 5 min and collect supernatant.
90 22 Estimation of Inorganic Phosphorus in Serum and Urine