The measurement of unbound calcium in serum

The measurement of the physiologically active form of the plasma calcium, the ionised fraction, presents severe technical problems. If non-protein bound calcium has to be separated from protein-bound calcium prior to analysis then artefactual dissociation of calcium proteinate might be minimised by the use of appropriate buffers, constant temperature environments and continuous flow automatic techniques. The latter techniques call for (a) a suitable mechanism to separate non-protein bound calcium from serum in a flowing stream ana (b) an appropriate means of detecting calcium continuously. The purpose of this work has been to explore the possibilities of fulfilling these two requirements. A review of the literature led to the conclusions that ionised and albumin bound calcium constitute approximately 85% of the total plasma calcium and that bound calcium is only loosely associated with plasma proteins. Also, no reasonable method is available at present for the measurement of non-protein bound calcium in the routine clinical chemistry laboratory. The possibility of using radioactive isotopes as tracers for calcium in a flowing stream was explored by examining the properties of commercially available flow cells. A new concept in continuous flow counting was also developed. The problem of separating non-protein bound calcium from serum in a flowing stream was approached in two ways. It was shown that gel filtration of serum resulted in the complete dissociation of calcium from proteins. However, separation was achieved using continuous flow dialysis. It was concluded that continuous flow dialysis may form the basis of a technique for the measurement of unbound serum calcium.