PRINCIPLES OF TRACER METHODOLOGY 9 



distribute itself in proportion to the stable form already present in the 

 system. For example, the calcium of blood exists in at least two forms: 

 diffusible calcium, which is considered to be ionized, and nondiflusiljle, 

 which is bound to a protein. The equilibrium may probably be repre- 

 sented by the following equation: 



Ca++ + Prot= ^ Ca Prot (1-1) 



Visek et al. (49) have shown that, if Ca*^ is added to plasma, the specific 

 activities of the ionized calcium and the protein-bound calcium become 

 equal within a matter of minutes. This supports the idea that the Ca 

 ions are in equilibrium in the two phases or at least are interchangeable. 



This situation leads to a very important application by means of which 

 a simple radioisotope measurement may yield the same information as a 

 difficult or often impracticable chemical analysis. It is clear that in the 

 above simple blood calcium system the relative proportions of ionizable 

 and bound calcium may be estimated by the addition of Ca^^ the separa- 

 tion of the two fractions, and the measurement of Ca*^ in each fraction. 

 To illustrate, assume that Ca"*^ is added to a serum sample which then 

 measures 1000 counts/min/0. 1 ml and that this sample is forced through a 

 cellophane or collodion membrane to give a solution containing only ion- 

 izable calcium which measures 400 counts/min/0. 1 ml. It is clear that 

 40 per cent of the original calcium was ionizable and 60 per cent was 

 bound. To estimate the absolute amounts of calcium in each fraction, 

 it would be necessary only to determine the total serum calcium in the 

 usual way: If the serum calcium w^ere 10 mg/100 ml, then the "ionizable" 

 and "bound" calcium values in the serum would be 4 and 6 mg/100 ml, 

 respectively. Small corrections would have to be made, in the usual 

 way, for the volume of protein, etc., but these would not invalidate the 

 general principles. This advantage is accentuated in dealing with more 

 complex systems. For example, Visek et al. (49) separated labeled blood 

 calcium into four fractions and demonstrated that the specific activities 

 were equal in all cases. In this separation it was difficult at times to get 

 enough sample for chemical analysis, but the counting of the Ca*^ added 

 to the system was always a simple matter and served to measure the 

 proportions of the original blood calcium in each fraction. 



A further example of how this principle might be applied is as follows: 

 The estimation of the natural cobalt content in animal tissues is difficult 

 because, except for the liver, the tissue content is very low and the 

 methods are generally tedious. If it could be shown that radiocobalt 

 administered to an animal reached an equal specific activity in all tissues, 

 then the natural cobalt content could be easily determined by a simple 

 radiocobalt assay on all tissues of interest plus a chemical cobalt deter- 

 mination on one tissue, presumably the fiver. Obviously this method 



