Introduction
- Phosphorus is an essential mineral present in the body mainly as phosphate.
- It is the second most abundant mineral after calcium.
- About 85% of body phosphorus is present in bones and teeth.
- The remaining phosphorus is present in soft tissues and extracellular fluid.
- In blood, phosphorus exists mainly as:
- inorganic phosphate
- organic phosphate esters
- Inorganic phosphate is the clinically important form measured in serum.
- Phosphorus is essential for:
- bone mineralization
- ATP formation
- nucleic acid synthesis
- phospholipid formation
- acid-base balance
- Serum phosphorus estimation is important for evaluating bone disorders, renal disease, parathyroid disorders, and vitamin D metabolism.
Principle
- Serum phosphorus estimation is based on UV molybdate method.
- Inorganic phosphate reacts with ammonium molybdate in acidic medium.
- A phosphomolybdic heteropolyacid complex is formed.
- Sulfuric acid eliminates the need for protein-free filtrate.
- The intensity of the complex formed is directly proportional to phosphorus concentration.
- Absorbance is measured at 340 nm.
Reaction
Phosphate + Ammonium→ Molybdate → Phosphomolybdic Complex
Specimen
Sample Type
- Serum free from hemolysis
- Urine may also be used after dilution
Precautions
- Use fresh non-hemolyzed sample
- Avoid contamination
- Separate serum early
Reagents
| Reagent | Composition | Concentration |
|---|---|---|
| Working Reagent | Ammonium Molybdate | 0.3 mmol/L |
| Sulfuric Acid | 1.0% | |
| Standard | Phosphorus Standard | 5 mg/dL |
Reagent Preparation
- Reagent and standard are ready to use.
Materials Required
- Test tubes
- Micropipette
- Pipette tips
- Spectrophotometer / colorimeter
- Cuvette
- Timer
- Phosphorus reagent kit
Procedure
| Components | Blank | Standard | Test |
|---|---|---|---|
| Reagent | 1000 µL | 1000 µL | 1000 µL |
| Standard | — | 10 µL | — |
| Sample | — | — | 10 µL |
Incubation
- Mix properly
- Incubate for 5 minutes at room temperature
Reading
- Measure absorbance against reagent blank
- Read at 340 nm
Calculation
Formula
Phosphorus (mg/dL) = Absorbance of Sample / Absorbance of Standard × Standard concentration
Normal Reference Values
| Group | Normal Value |
|---|---|
| Adult | 3.0 – 4.5 mg/dL |
| Children | 4.0 – 5.5 mg/dL |
Clinical Significance
Increased Serum Phosphorus (Hyperphosphatemia)
- Renal failure
- Hypoparathyroidism
- Vitamin D excess
- Bone destruction
Decreased Serum Phosphorus (Hypophosphatemia)
- Hyperparathyroidism
- Vitamin D deficiency
- Malabsorption
- Rickets
Diagnostic Importance
- Assesses bone metabolism
- Evaluates renal function
- Helps diagnose parathyroid disorders
