Introduction
- The Inorganic Phosphate (Pi) test is a laboratory investigation used to measure the concentration of inorganic phosphate in blood serum or plasma.
- Phosphate is an essential mineral required for various physiological and biochemical functions in the body.
- Approximately 85% of body phosphate is present in bones and teeth, while the remaining amount is distributed in soft tissues and body fluids.
- Phosphate plays an important role in:
- Bone and teeth formation
- Energy production (ATP synthesis)
- Nucleic acid synthesis (DNA and RNA)
- Cell membrane structure
- Acid–base balance
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.