Introduction
- Calcium is an essential mineral in the body, playing a critical role in various physiological processes, including muscle contraction, nerve function, blood clotting, and bone metabolism.
- Determining calcium levels in serum, plasma, or urine is vital for diagnosing and managing hypercalcemia, hypocalcemia, parathyroid gland disorders, kidney disease, and bone-related disorders like osteoporosis.
Principle
The calcium determination is commonly based on the reaction of calcium ions with specific colorimetric reagents (e.g., o-cresolphthalein complexone or Arsenazo III). These reagents form a colored complex with calcium, and the intensity of the color, measured spectrophotometrically, is directly proportional to the calcium concentration.
The reaction occurs in an alkaline medium, and interference from magnesium and other ions is minimized by adding chelating agents like 8-hydroxyquinoline.
Methods
- Colorimetric Method (o-Cresolphthalein Complexone)
Samples
- Sample Type:
- Serum, plasma (heparinized), or urine. Avoid using EDTA, citrate, or oxalate anticoagulants as they chelate calcium and falsely lower levels.
- Sample Volume: ~0.5 mL.
- Collection Notes:
- Fasting samples are preferred as calcium levels may fluctuate with food intake.
- Storage:
- Analyze fresh samples within 24 hours. Store at 2–8°C for the short term or freeze for longer storage.
Reagents
- Calcium reagent l: lt is prepared by mixing 40 mg of cresolpthalein complexon in 1.0 ml of conc. hydrochloric acid, followed by 2.5g of 8-hydroxyquinoline, 100 ml of dimethyl sulfoxide, and the final quantity is made up to 1 liter using glass distilled water.
- Calcium reagent 2: It is prepared by mixing 500 mg of potassium cyanide and 40 ml of diethylamine in 960 ml of glass distilled water.
- Calcium standard: 10 mgldl
Procedure
| Test | Std. | Blank | |
| Working calcium reagents, ml | 1.0 | 1.0 | 1.0 |
| Serum, ml | 0.01 | – | – |
| Std. ml | – | 0.01 | – |
| Blank | – | – | 0.01 |
Mix and keep at room temp for 10 min. Read the absorbance of the test and standard and blank at 575nm (Yellow filter).
Calculation
Determine the calcium concentration from the standard curve:
Calcium (mg/dL) = Absorbance of sample/Absorbance of standard×Concentration of standard (mg/dL)
Calcium excretion can be expressed in mg/day based on urine volume for urine samples.
Normal Range
- Serum Calcium:
- Adults: 8.5–10.5 mg/dL
- Children: 9.0–11.0 mg/dL
- Ionized Calcium: 4.6–5.3 mg/dL
- Urine Calcium (24-hour):
- Adults: 100–300 mg/day
Clinical Significance
- Increased Calcium (Hypercalcemia):
- Hyperparathyroidism
- Malignancy (e.g., bone metastases, multiple myeloma)
- Vitamin D intoxication
- Granulomatous diseases (e.g., sarcoidosis)
- Prolonged immobilization
- Decreased Calcium (Hypocalcemia):
- Hypoparathyroidism
- Vitamin D deficiency
- Chronic kidney disease
- Hypoalbuminemia (affects total calcium but not ionized calcium)
- Acute pancreatitis