Introduction
- Inorganic phosphate (Pi) is vital in numerous physiological processes, including energy metabolism, bone mineralization, and acid-base balance.
- It is measured to assess renal function, parathyroid gland activity, and bone health.
- Inorganic phosphate levels in blood or urine can help diagnose hypophosphatemia, hyperphosphatemia, kidney disorders, and hormonal imbalances.
Principle
Inorganic phosphate reacts with ammonium molybdate in an acidic medium to form phosphomolybdic acid. This complex is then reduced by a reducing agent (e.g., stannous chloride) to produce a blue-colored compound, molybdenum blue, the intensity of which is proportional to the concentration of phosphate. The absorbance is measured spectrophotometrically at a wavelength of 660–700 nm.
Methods
Gomorri’s method
Samples
- Sample Type:
- Serum, plasma (heparinized), or urine.
- Sample Volume: ~0.5 mL.
- Collection Notes: Avoid hemolysis, as intracellular phosphate from red blood cells can falsely elevate levels.
- Storage:
- Analyze samples within 24 hours. Store at 2–8°C for short-term preservation.
Reagents
- 10g/dl, trichloroacetic acid acid in distilled water
- Molybdate reagent: It is prepared by dissolving 0.5 g of ammonium molybdate in 100 ml of 10 N sulfuric acid and 300 ml of distilled water.
- Colour reagent: It is prepared by dissolving 1.0 g of metol in 100 ml of 3 g/dl sodium metabisulfite solution.
- Phosphate standard solution: 5.0mg/dl
Procedure
| Test | Diluted Std | |
| TCA reagent, ml | 4.5 | 4.5 |
| Serum, ml | 0.5 | – |
| Standard, ml | – | 0.5 |
Mix, centrifuge, test, and get clear filtrate.
| Test | Std. | Blank | |
| Filtrate, ml | 2.5 | – | – |
| Diluted Std., ml | – | 2.5 | – |
| Distilled water, ml | – | – | 2.5 |
| Molybdate reagent, ml | 0.5 | 0.5 | 0.5 |
| Color reagent | 0.5 | 0.5 | 0.5 |
Mix thoroughly and keep in the dark for 10 min. Measure the absorbance at 660–700 nm (Red filter)
Calculation
Determine the phosphate concentration from the standard curve:
Concentration of phosphate (mg/dL) = Absorbance of sample/Absorbance of standard×Concentration of standard (mg/dL)
Normal Range
- Serum/Plasma:
- Adults: 2.5–4.5 mg/dL
- Children: 4.0–7.0 mg/dL (higher due to active bone growth)
- Urine (24-hour): 400–1,300 mg/day
Clinical Significance
- Increased Inorganic Phosphate (Hyperphosphatemia):
- Chronic kidney disease (impaired phosphate excretion).
- Hypoparathyroidism.
- Excess vitamin D or phosphate intake.
- Bone diseases (e.g., Paget’s disease).
- Decreased Inorganic Phosphate (Hypophosphatemia):
- Malnutrition or malabsorption.
- Hyperparathyroidism.
- Vitamin D deficiency.
- Refeeding syndrome.
- Chronic alcohol abuse.