Human biomarkers for measuring calcium intake and status

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).

It is possible to measure ionised (free) calcium only. Measurements of ionised calcium are considered more clinically relevant than total calcium, this is because it is the physiologically active form and unlike total calcium it is not affected by fluctuations in albumin concentration (22).

Ionised calcium in whole blood is measured by ion-selective electrode on specialised electrolyte or blood gas chemistry analysers (e.g. from Novamedical or Radiometer). In a clinical setting, it is more common to measure ionised calcium using a blood gas analyser at the point of care. Ionised calcium can be measured in heparinised whole blood. Blood should be drawn anaerobically, mixed per protocol, placed on ice and measured with 30 min. Changes in pH have a significant effect on ionised calcium and samples should be measured soon after collection. As the collection protocol for ionised calcium is very restrictive and does not allow for the use of stored samples it is often more practical to measure total calcium.

Parathyroid hormone (PTH) and 1,25(OH)2D are elevated when there is increased calcium demand or when calcium intakes are low (27). Calcitonin also has a role in maintaining serum calcium (28).

Bone density measurements (e.g. by dual X-ray absorptiometry, computerized tomography or quantitative ultrasound) may be used as long-term indicators of the adequacy of calcium intake, and are often combined with dietary assessment of calcium intake (assessed via questionnaire)(16).

Bone turnover markers (e.g. bone-specific alkaline phosphatase or osteocalcin) may also provide insight into relative calcium requirements. Metabolic adaptations to pregnancy and lactation will affect interpretation of calcium and bone-related biomarkers (29).

Several further novel metabolites from urinary metabonomics analysis of calcium-deficient and normal rats have been proposed as biomarkers of calcium deficiency (30)but these have yet to be validated as indicators of calcium status of use for human population assessment.