Geyer, Robert & Bowie, E.‘The Direct Microdetermination of Tissue Calcium
by Flame Photometry’,Anal Biochem, 2, (1961), p. 360-369.
A number of methods have been reported for the measurement of calcium in biological materials by flame photometry or titration.
This method employed trichloroacetic acid (TCA)
to precipitate the proteins and had been found to be
adequate to extract all of the calcium from tissue samples. Sodium, potassium and phosphate caused no significant interference to calcium concentration in the range of 0 to 2 µg/ml and was readily determined.
Solutions
Deionised distilled water was used throughout. Diluting Solutions was prepared from 500ml isopropyl alcohol, 300ml deionised water, 0.2ml Non-Ion-Ox (Aloe Scientific Co, St. Louis, Mo.), 74.6 mg KCl and 292 mg NaCl. Trichloroacetic acid solution was 6.2% w/v for tissue extraction.
Procedure
Tissue samples were prepared and carefully blotted on filter paper, then accurately weighed to yield samples of in the range of 100mg wet weight.
The sample was transferred to a small centrifuge tube and sufficient 6.2% TCA solutions was added to bring the volume to 2 ml. The tube was tightly covered and vigorously shaken three times over a thirty-minute period in ten-minute intervals.
A volume of 8 ml of Diluting Fluid was added to the centrifuge tube and the tube was centrifuged. The supernatant solution was analysed directly without further dilution.
When measuring samples, an initial test to estimate the range of calcium concentration via a single point calibration of 100ppm was be carried out. With the estimated value of sample concentration, standards should then be made up of at least 4 standards covering ±50ppm from the rough guide sample value.
When producing standard solutions, it was of vital importance that they were diluted in the Diluting Solution and the Diluting Solution should be ran as a blank so that the flame photometer accounts for chemical interferences in the matrix as well as the influence of isopropyl alcohol being present in the flame.
Normal physiological concentrations of sodium, potassium and phosphate were not found to cause significant interferences. However, methods for reducing the chemical interference found from these could be avoided by methods utilising EDTA, found in Measurement of Calcium in Serum and Urine. Phosphate interference can also be reduced by increasing flame temperature and methods have been proposed that the addition of trace quantities of Lanthanum and Strontium, however the extent by which the interference is reduced is not currently quantified.