Introduction
- Calcium is one of the most important minerals in the human body.
- About 99% of total body calcium is present in bones and teeth.
- The remaining calcium is present in extracellular fluid and intracellular compartments.
- Serum calcium plays an essential role in neuromuscular function, blood coagulation, enzyme activity, hormone secretion, and membrane stability.
- Calcium exists in serum in three forms:
- ionized calcium (physiologically active form)
- protein-bound calcium
- calcium complexed with anions
- Serum calcium estimation is an important biochemical test used to assess bone metabolism, parathyroid function, renal disease, and metabolic disorders.
Principle
- Serum calcium estimation is based on Arsenazo III method.
- Arsenazo III dye combines with calcium ions in acidic medium to form a blue-purple colored complex.
- The intensity of color formed is directly proportional to calcium concentration in the sample.
- Absorbance is measured at 650 nm.
Reaction
Calcium + Arsenazo III → Calcium-Arsenazo III colored complex
- Arsenazo III has high specificity for calcium and shows minimal interference from other serum ions.
Specimen
Sample Type
- Serum is preferred specimen
- Heparin plasma can also be used
- Urine may also be analyzed
Precautions
- Use non-hemolyzed sample
- Avoid calcium contamination
- Plastic tubes are preferred
Stability
Serum / Plasma
- 7 days at 20–25°C
- 3 weeks at 4–8°C
- 8 months at −20°C
Reagents
Reagent 1
- Arsenazo III
- Phosphate buffer (pH 7.8)
Standard
- Calcium standard solution
Reagent Preparation
- Reagents are liquid and ready to use
Materials Required
- Test tubes
- Micropipette
- Pipette tips
- Colorimeter / spectrophotometer
- Cuvette
- Timer
- Calcium reagent kit
Procedure
| Components | Blank | Standard | Test |
|---|---|---|---|
| Reagent 1 | 1000 µL | 1000 µL | 1000 µL |
| Distilled water | 10 µL | — | — |
| Standard | — | 10 µL | — |
| Sample | — | — | 10 µL |
Incubation
- Mix properly
- Incubate at 37°C for 1 minute
Reading
- Measure absorbance of test and standard against blank
- Read at 650 nm
Calculation
Formula
Calcium (mg/dL) = Absorbance of Test / Absorbance of Standard × Standard concentration
Unit Conversion
mg/dL × 0.25 = mmol/L
Normal Reference Values
| Group | Normal Value |
|---|---|
| Adult | 8.6 – 10.2 mg/dL |
| Child (2–12 years) | 8.8 – 10.8 mg/dL |
Clinical Significance
Increased Serum Calcium (Hypercalcemia)
- Hypercalcemia means increased serum calcium above normal range.
- It usually indicates increased bone resorption, excessive intestinal absorption, or reduced renal excretion.
Seen in:
- Hyperparathyroidism
- Paget disease of bone
- Malignancy with bone metastasis
- Vitamin D intoxication
- Prolonged immobilization
Clinical Effects
- muscle weakness
- constipation
- polyuria
- renal stones
- cardiac arrhythmias
Hyperparathyroidism
- Excess parathyroid hormone increases bone calcium release.
- Renal calcium reabsorption also increases.
- Serum calcium becomes elevated.
Malignancy
- Some cancers produce hypercalcemia by bone destruction or tumor-related humoral factors.
Decreased Serum Calcium (Hypocalcemia)
- Hypocalcemia means decreased serum calcium below normal range.
- It often occurs due to reduced absorption, reduced parathyroid hormone activity, or vitamin D deficiency.
Seen in:
- Rickets
- Osteomalacia
- Hypoparathyroidism
- Chronic renal disease
- Malabsorption syndrome
Clinical Effects
- tetany
- muscle cramps
- paresthesia
- seizures
Rickets and Osteomalacia
- Calcium deficiency impairs bone mineralization.
- Serum calcium may be reduced.
Renal Disease
- Kidney disease reduces vitamin D activation and calcium balance.
Diagnostic Importance
- Helps assess bone disorders
- Useful in parathyroid disease diagnosis
- Important in renal disease evaluation
- Monitors electrolyte balance
