Calcium : Introduction, clinical significance and measurement

About 99% of calcium occurs in bone, along with calcium 85% of phosphate and 55% magnesium. The concentration of these minerals depends on net effect of bone mineralization, intestinal absorption and renal excretion. PTH and 1, 25-dihydroxyvitamin D are the principle hormones regulating these three processes.

Calcium is the 5^th most abundant element, and most prevalent cation. Average human body contains approximately 1 kg of calcium. It is found in skeleton (99%) as hydroxyapatite crystal, soft tissues (1%), and extracellular fluid (<0.2%).

In blood virtually all of the calcium is in plasma, with normal concentration of 9.5 mg/dl (2.38 mmol/L). 50% is free (ionized) which is active form, 40% is protein bound mainly albumin followed by globulin and 10% is complexed with small anions like lactate, phosphate, bicarbonate and citrate. The concentration of free form is regulated by calcium regulating hormones PTH and 1, 25-dihydroxyvitamin D. It binds to negatively charged proteins so binding is pH dependent more binding occurring in alkaline pH. In multiple myeloma high concentration of globulin may bind calcium reducing free pool.

The skeleton is a major reservoir for providing calcium for both extracellular and intracellular pools. Intracellularly calcium is involved muscle contraction, release of granules in hormone secretion, cell division, etc. Extracellular calcium provides calcium for maintenance of intracellular calcium, bone mineralization, blood coagulation, and plasma membrane potential.

Calcium is absorbed from GI by two mechanisms one is active transport involving vitamin D, calcitriol in duodenum and jejunum and another is by passive method in colon. The absorption of calcium is influenced by dietary constituents. The presence of anions such as phosphate, oxalate (in green vegetables), and phytate (in cereals) diminishes calcium solubility and thus absorption.

CLINICAL SIGNIFICANCE

Hypocalcemia

Fig. Hypocalcemia signs

This may be due to reduced albumin bound calcium, free calcium or both. Hypoalbuminemia is the cause of pseudohypocalcemia. Other conditions resulting to hypocalcemia are chronic liver disease, nephrotic syndrome, congestive heart failure, and malnutrition, osteomalacia and rickets, etc. but common causes are chronic renal failure and hypomagnesemia. In chronic renal failure, hypoproteinemia, hyperphosphatemia, low serum vitamin D (reduced synthesis because of inadequate renal mass) and/or skeletal resistance to PTH contribute to hypocalcemia. Magnesium deficiency impairs PTH secretion and cause PTH end-organ resistance. Inherited resistance of PTH leads to pseudohypoparathyroidism and thus hypocalcemia e.g. in pseudohypoparathyroidism type I (Albright’s hereditary osetodystrophy) is due to reduction in guanine nucleotide regulatory complex in adenylate cyclase complex. Vitamin D deficiency is also associated with hypocalcemia and is due to impaired intestinal absorption of calcium and skeletal resistance to PTH. Clinically hypocalcemia presents with neuromuscular hyperexcitability, such as tetany, paresthesia, and seizures.

Initial lab assessment is measurement of renal function and measurement of serum albumin and magnesium concentrations. Other parameters are low vitamin D, PTH or sometimes high PTH due to resistance, and high serum ALP. Large amounts of burn cream contain polyethylene glycols which are absorbed and metabolized to dicarboxylic acid that bind calcium. Patient develops elevated total calcium but low free calcium, along with metabolic acidosis and increased serum osmolality from glycols.

Hypercalcemia

This occurs due to excessive bone resorption like in malignancy. Failure of kidney to excrete filtered calcium also caused hypercalcemia. It may be caused by increased intestinal absorption (vitamin D intoxication), increased renal retention (thiazide diuretics), increased skeletal resorption, or combination of mechanisms (primary hyperparathyroidism which occur due to adenoma, hyperplasia, etc.) is the most common cause of hypercalcemia (90%) in outpatients and malignancy (95%) in inpatients. Primary hyperPTH is characterized by excessive secretion of PTH causing hypercalcemia. The most common symptoms are fatigue, malaise, weakness and mild hypercalcemia, etc. Increase in albumin or globulins as in multiple myeloma binds more calcium and cause to increase total calcium. Hypercalcemia causes hypercalciuria which can lead to renal calculi. In sarcoidosis and other granulomatous disease tissue contains 25-hydroxyvitamin D-1α-hydroxylase required to produce active vitamin D.

Fig. Causes of Hypercalcemia

Laboratory analysis includes measurement of serum calcium (ideally free calcium), albumin, PTH, 1, 25 vitamin D.  

Hypercalcemia affects from 0.1 to 1% of the population. The widespread ability to measure blood calcium since the 1960s has improved detection of the condition, and today most patients with hypercalcemia have no symptoms. Women over the age of 50 are most likely to be hypercalcemic, usually due to primary hyperparathyroidism.

MEASUREMENT OF CALCIUM

Measurement of calcium includes either free or total calcium. The term ionized calcium is misnomer because all plasma calcium is ionized either free or bound form so, free form is appropriate. Free calcium is the best indicator of calcium status as it is the active form and tightly regulated by PTH and vitamin D. ISE and other autoanalyzers are available to measure free but preferably total calcium.  Corrected calcium is often used that corrects measured calcium with albumin.

Corrected total calcium (mg/dL) = total calcium (mg/dl) + 0.84 (4-albumin [g/dl])

; 4 represents the average albumin level in g/dL.

In other words, each 1 g/dL decrease of albumin will decrease 0.8 mg/dL in measured serum Ca and thus 0.8 must be added to the measured Calcium to get a corrected Calcium value.

Factors affecting protein binding to calcium includes, altered albumin or globulins, heparin, pH, free fatty acids, bilirubin, etc. Whereas factors altering complex formation includes, citrate, bicarbonate, lactate, pyruvate, sulfate, anion gap, etc.

The methods of total calcium measurements includes

Photometric methods

i.     O-Cresolphthalein complexon method

In alkaline solution, the metal-complexing dye CPC forms a red chromophore with calcium measured at 580 nm. The sample is diluted with acid to release protein-bound and complexed calcium. Use of Hydroxyquinolone, pH 12 and measuring at 580 nm are used to prevent interference from magnesium. Diethylamine is used to produce alkaline pH.

ii.   Arsenazo III method

Arsenazo III at mild acidic condition (pH 6) has higher affinity to calcium than magnesium. Imidazole is used to buffer the reaction. Interference from most biological pigments is reduced by measuring the complex at 650 nm.

iii. Clark and collip method

Serum total calcium is precipitated as calcium oxalate, which is washed with ammonia and dissolved in acid. Oxalic acid thus formed is titrated at 70⁰C- 80⁰C against standard permanganate. From the titre value the calcium content of serum is calculated.

Calcium + ammonium oxalate -------> calcium oxalate (ppt)

2KMNO4 + 3H₂SO₄ + 5(COOH)₂ -----> K₂SO₄ + 2MnSO₄ + 8H₂O + 10CO₂

Atomic Absorption Spectrometry(AAS) method

Use of AAS is the reference method for measuring total serum calcium and IDMS is the definitive method. In this method, the specimen is first diluted with lanthanum-HCL, and then aspirated into an air-acetylene flame, where the ground state calcium atoms absorb incident light from a calcium hollow cathode lamp (422.7 nm). The amount of light absorbed is measured by phototube or detector after the 422.7 nm resonance line is isolated with the monochromator. Absorbance is directly proportional to the number of ground state calcium atoms in the flame.

Dilution with lanthanum-HCl reduces interference from protein, phosphate, citrate, sulfate and other anions. Phosphate causes the greatest interference because calcium phosphate complexes are not dissociated readily by air-acetylene flame. Lanthanum-HCl dissociates complexes ensuring that all fractions of calcium are measured. Dilution reduces viscosity which improves aspiration rate.

Specimen requirement

Serum and heparinized plasma are the preferred specimens. Hemolysis, icterus, lipemia, paraproteins, Hb, bilirubin and Mg interfere with the test.

Measurement of free (Ionized) calcium

These used ISE that determine free calcium from whole blood. The instrument contains calcium ion-selective, reference, and pH electrodes. Sensitive potentiometers measure the voltage difference between the calcium or pH and reference electrodes for calibrating solutions or samples. Calcium ISE contains a calcium selective membrane, which encloses an inner reference solution of calcium chloride often containing saturated AgCl and physiological concentration of NaCl and KCl and an internal reference electrode. The reference electrode usually of Ag/AgCl is immersed in this inner reference solution. The electrochemical systems include reference electrodes, calcium sensitive electrode and salt bridge like in pH electrode. The potential difference across the cell is logarithmically related to the activity of free calcium ions in sample by Nernst’s equation.

Specimen requirement for ISE

Heparinized whole blood, heparinized plasma or serum can be used. CO₂ loss should be prevented otherwise this will increase in pH as occurs when specimen are exposed to air. Ideally whole blood specimen should be analyzed within 15 to 30 minutes of sampling. But 1 hr at room temperature and 4 hr at 4⁰C is reported to be stable. Serum specimens shows greater stability. The practice of using aerobic specimens for measurement of free calcium should be abandoned.

PATIENT PREPARATION FOR CALCIUM MEASUREMENT

Due to tourniquet application total calcium is altered but not free calcium and this is due to venous occlusion and there is efflux of water from the vascular compartment during stasis. Fist clenching or other forearm exercise should be avoided before phlebotomy, because forearm exercise causes decrease in pH (lactic acid production) and an increase in free calcium. Standing decreases intravascular water and increase the total calcium. Prolonged immobilization and bed rest can decrease bone density and increase total and free calcium.

REFERENCE INTERVALS

Total calcium = 8.6-10.2 mg/dL (2.15 – 2.55 mmol/L)

Free calcium = 4.6 -5.3 mg/dL (1.15 – 1.33 mmol/L)

Since free calcium is affected by pH, it is recommended that pH be measured and reported with all free calcium determinations.

Free calcium is more useful than total calcium determination in hospital patients, especially those undergoing major surgery who have received citrated blood or platelets, heparin, bicarbonate. Rapid measurement of free calcium, blood gases, and potassium permits maintenance of good cardiac function during surgery. Free calcium is more useful than total in diagnosis of hypercalcemia as in primary hyperparathyroidism where there is increase in free calcium than total and also malignancy.

Measurement of calcium in urine reflects intestinal absorption, skeletal resorption and renal tubular filtration and reabsorption. The measurement is useful in assessing renal stone disease and high-turnover osteoporosis. Calcium oxalate crystals can be seen in case of stones. Acidification of urine is done by 6 mol/L HCl to prevent calcium salt precipitation.