Geyer, R.P. and Bowie, E.J., ‘The Direct Microdetermination of Tissue Calcium by Flame Photometry’, Anal. Biochem., 2, (1961), 360-369.
This procedure uses a TCA (trichloroacetic acid) extraction of the tissue to release all of the Calcium
Procedure
Solution and Standard Preparation
Deionised distilled water was used throughout to prepare the following solutions:
Organic Diluting Fluid contained 500ml isopropyl alcohol, 300 ml water, 0.2 ml Non-Ion-Ox (ex. Aloe Scientific Co., St. Louis, Mo. USA), 74.6 mg KCl and 292 mg NaCl.
TCA solution for tissue extraction was 6.2% w/v and for standard preparation was 8.3% w/v and also contained 19.5 mg NaCl / 100ml.
6.25% trichloroacetic acid solution was first prepared. 6.25grams of TCA acid was accurately weighed and added to a 100ml volumetric flask and was then made up to the mark with deionised or MilliQ water. An 8.3% solution was then made up using 8.3grams of TCA into 100ml of DI water in a volumetric flask.
Primary Calcium Standard Solution was prepared by dissolving 250 mg CaCO3 in 25ml of 2M HCl and diluted to 1l to give 100 μg Ca / ml
Standards and reagent blanks containing 0 – 3 μg Ca /ml were prepared by mixing 80ml of Organic Diluting Fluid, 15ml of 8.3% TCA, 0 – 3ml of Primary Calcium Solution and made up to the mark with DI water. To this solution 1gram of Lanthanum Chloride was added. The 0 μg Ca /ml solution was used as a blank solution and then calibrated in increasing amounts of concentrations (1, 2 then 3 μg Ca /ml).
Tissue Extraction
A tissue slice of approximately 0.5mm thickness was prepared and then quickly dipped into a calcium free incubation solution. This was then blotted carefully on a piece of filter paper. The tissue was then accurately weighed to yield samples of approximately 100mg wet weight.
The sample was transferred to a centrifuge tube and adequate 6.2% TCA solution was added to bring the total volume of the sample to 2ml. This solution was then vigorously shaken every ten minutes over a half hour period. 8ml of Organic Diluting fluid was added to the tube and then the solution was centrifuged at 2000g for 30 minutes. The supernatant fluid was then pipetted off and 0.1grams of Lanthanum Chloride was added and mixed well. This solution was then aspirated
through the pre-calibrated flame photometer.
Preparation of Standard Graph
Set the flame photometer in accordance to MultiPoint/Single Ion Calibration found on page 24 of the BWB Technologies Installation and Operation Manual, to measure calcium emission.