MAKE BIOCHEMISTRY MANUAL

Q1. Write down the method and procedure of Serum Calcium estimation (Total ionized
and unionized). Also write down the reference range.

SERUM CALCIUM ESTIMATE:

INTRODUCTION:
Calcium is one of the most important and plentiful mineral nutrients for the human body.
Approximately 98-99% is present in the bones and calcified tissues and a small amount about 1-
2% is found in extracellular fluids.

Serum calcium determination is useful in monitoring certain clinical conditions such as bone
related disorders, acid – base balance, renal failure etc.

ESTIMATION OF SERUM CALCIUM:

NAME OF METHOD

O – CRESOLPHTHALEIN COMPLEXONE METHOD (OCPC METHOD)

PRINCIPLE:
Calcium combines with o- cresolphthalein complexone in alkaline medium to form a purple
colored complex. This reagent also contains 8 – hydroxyquinoline which minimize the
interference of magnesium. Intensity of developed color is directly proportional to the
concentration of calcium present in the serum.

1. Calcium +o-Cresolphthalein complexone alkaline pH Purple colored complex

2. 8- hydroxyquinoline

REQUIREMENTS:
Test tubes, Test tube stand, serological Pipettes, water bath or incubator, colorimeter, reagent test
kit etc.

REAGENTS:
The estimation kit has three reagents

(i) Reagent 1: Buffer solution

(ii) Reagent 2: Color reagent

(iii) Reagent 3: Standard reagent

PROCEDURE:
• 1. Take a test tube stand and placed three clean and dry test tubes.
• 2. Label the tubes as test, standard and blank. Pipette in the labelled tubes as follows:

• 3. Mix well and keep at room temperature for 5 minutes.

• 4. Measure the color intensity (OD) of test and standard against blank at 570 nm.

CALCULATION:
 Serum Calcium, mg/dl = OD of Test x Concentration of standard reagent
OD of Standard

NORMAL VALUE:
• Serum Calcium, mg/dl: 8.5 – 11.0 mg/dl

CLINICAL SIGNIFICANCE:
• Hypercalcemia may develop in patients with Paget’s disease of bone,
hyperparathyroidism, malignancy of bone, hypervitaminosis D, multiple myeloma etc.
• Hypocalcemia found in conditions such as rickets, osteo-malacia, low plasma protein
concentration, vitamin D deficiency, hypoparathyroidism and renal failure.
The normal value of calcium in serum is 9–11 mg/dl and 100–300 mg/24 h urine. Extracellular
calcium provides calcium ions for maintenance of intracellular calcium, bone mineralization,
blood coagulation, and maintenance of plasma 21.8 Clinical Significance 87 membrane potential.
Calcium levels are increased in hyperparathyroidism (determination of ionized serum calcium is
more useful for the diagnosis of hyperparathyroidism), parathyroid hormone injection,
hypervitaminosis “D,” prolonged intake of milk and alkali (the milk-alkali syndrome), multiple
myeloma, and polycythemia. It is also increased in acute and chronic renal failure and
osteomalacia with malabsorption. Hypocalcemia occurs when serum calcium levels fall below 7
mg/dl. Hypocalcemia is more serious and life-threatening condition than hypercalcemia. Calcium
levels are decreased in hypoparathyroidism, osteomalacia, hyperphosphatemia, tetany, renal
failure, and nephrotic syndrome. In rickets, the product of serum calcium and phosphorus
decreases usually below 30 mg/dl. An increase in alkaline phosphatase activity is a characteristic
feature of rickets. It is necessary to measure total serum proteins and albumin levels
simultaneously for proper interpretation of serum calcium levels, since 0.8 mg of calcium is
bound to 1.0 g of albumin in serum. To correct, add 0.8 mg/dl for every 1 g/dl that serum
albumin falls below 4 g/dl.