PRINCIPLES OF TRACER METHODOLOGY 47 



analysis of amino acids in mixtures. In general, the procedure is as fol- 

 lows: (a) The unknown acids are converted to labeled derivatives by 

 reaction with I'^'-pipsyl chloride, (b) A pure sample of each amino acid 

 to be measured is labeled with S'^^-pipsyl chloride, and a known amount 

 of each derivative is added to the unknown mixture, (c) The mixture is 

 separated into components on a paper chromatogram. (d) The bands of 

 the paper chromatogram are sectioned, the amino acids eluted from the 

 sections, and the S'^VI'" ratios determined. The identity of the bands 

 is determined from previous experience with the movement of known 

 amino acids. If a given band is pure, this will be shown by a constant 

 g35/ji3i j,jj^Jq Jj^ ^}^g various sections of the band. Furthermore the orig- 

 inal content of the I'^' derivative of a particular amino acid can be quan- 

 titatively estimated from the S^VI^^' ratio by the same type of calculation 

 as described above for the iron experiments. 



MISCELLANEOUS APPLICATIONS 



The investigator who thoroughly understands the basic principles of 

 tracer methodology and considers them in terms of his specific problems 

 is in an excellent position to develop novel and useful applications. Fol- 

 lowing are a few illustrations: 



Bioassays. The various techniques available for the assay of vita- 

 min D are not considered entirely satisfactory. Snyder, Eisner, and 

 Steenbock (106) have proposed a method that apparently offers many 

 advantages. It is based upon the effect of the vitamin D status of a test 

 animal upon the uptake of P^'- as determined by an external measurement. 

 Young rats are fed upon a rachitogenic ration for 16 days and are then 

 given orally an unknown vitamin D preparation in a potency range of 

 }4 to 50 units. After 48 hr the rats are given an intraperitoneal injection 

 of P^-, and following 10 days of continued feeding of the rachitogenic 

 ration the animals are anesthetized and an external count taken of the 

 forepaw. A standard reference curve is prepared by the identical treat- 

 ment of rats receiving doses of known vitamin D potency. 



The increase in P''*' excretion following the administration of para- 

 thyroid hormone to thyroparathyroidectomized rats has been used as a 

 basis for the bioassay of parathyroid hormone by Rubin and Dorfman 

 (107). In principle, the hormone and P^- in 1.2 mg disodium phosphate 

 were administered about 24 hr after the operation, and the urine then 

 collected for radioassay. Amounts as small as 0.5 USP unit were detect- 

 able; this represents a 200-fold increase in sensitivity over the standard 

 USP method. Also the need for tedious chemical estimations of blood 

 phosphate was eliminated. 



