Serum calciumexists in three forms: i) free calcium ions, Ca2+, at about 50% of the total, ii) protein bound calcium, at 45% of the total, and iii) complexed calcium, mainly with citrate, at about 5% of the total. The ionised calcium is physiologically most significant and indicates disturbances in calcium metabolism.
Total calcium can be measured in serum or lithium heparin plasma. Calcium cannot be measured in EDTA plasma and anticoagulants other than lithium heparin should be avoided because of complexing or precipitating the calcium. Urine calcium is also commonly measured.
Total calcium in serum/plasma or urine, is routinely measured using spectrophotometry on a clinical chemistry analyser; reagent kits are supplied ready to use and the most common methods are the o-Cresolphthalein Complexone method and the Arsenazo-III method.Serum/plasma total calcium concentration is stable for 7 days at room temperature (15-25°C), about 22 days under refrigeration (2-8°C) and for longer than 1 year frozen at -20°C, provided no sample evaporation has occurred. Calcium was found to be stable in serum, for ≤3 freeze-thaw cycles at -20°C (21).
Because a large fraction of circulating calcium is bound to albumin, fluctuations in albumin concentration may have a profound effect on serum/plasma calcium concentration. Therefore, adjustment for albumin may be considered (22).Albumin is reduced in malnutrition and is also a negative acute phase marker (i.e. is reduced in infection), which can affect total calcium concentrations.
Acidification of urine samples is often recommended for the assessment of urinary calcium (and other minerals) to prevent precipitation and formation of calcium crystals. Whilst recommended, recent data suggest that acidification may not be essential in a clinical setting (23, 24). Acid may be added to the collection bottles or laboratory aliquots at a concentration of 10 ml/L of hydrochloric acid. Contamination needs to be carefully avoided by using new or acid-washed collection vessels.
Urinary calcium is stable for 5 days at room temperature (15-25°C), for 5 weeks under refrigeration (2-8 °C) and for 6 months frozen at temperatures up to -20°C (25). Long term storage for 15 y at -22°C did not affect calcium concentrations in urine (26).