382 RADIOISOTOPES IN BIOLOGY AND AGRICULTURE 



tion. On the basis of these studies, a standardized quantitative proce- 

 dure was developed. 



Paper chromatography used in conjunction with I^^^ has been of partic- 

 ular value in the study of the various closely related iodine compounds 

 involved in animal metabolism, as illustrated in a series of papers by 

 Chaikoff and associates (-46 to 49) . The general procedure employed was 

 as follows (46) : Strips of Whatman No. 1 filter paper 10 by 36 cm were 

 employed. The butanol extract, containing the substances to be sep- 

 arated, was added in a thin band, 5 cm long, at 2.5 cm from the edge of the 

 paper. Up to 250 n\ of extract was added in successive 25-/xl applications, 

 each being allowed to dry before the next was added. The paper strips 

 were suspended in cylindrical pyrex jars (18 in. high and 8}-^ in. in diam- 

 eter) containing 150 ml of solvent. The strips were hung from a rack 

 attached to plate glass (which also served as a cover for the jar), with the 

 bottom of the strip dipping into the solvent. The solvent was prepared 

 by the addition of 44 ml water to 125 ml of redistilled collidine. A small 

 beaker of concentrated NH4OH was placed in the jar. The chromato- 

 gram was allowed to develop for 16 hr at 25 to 26°C. When I^^^ was used, 

 the dried chromatogram was placed in contact with X-ray film to give an 

 autoradiogram. When known stable compounds were used for reference 

 purposes, the dried chromatogram was sprayed with 2.5 per cent Na2C03 

 and then an aqueous solution of sulfanilic diazonium chloride to give the 

 colored bands. 



Figure 8-8 (49) illustrates some of the results obtained in regard to the 

 enterohepatic circulation of thyroxine and demonstrates not only the 

 resolution of a mixture but also the isolation of a pure substance to be 

 used for further study. The autoradiogram on the left represents the 

 bile from a rat that had been injected more than 3 hr previously with P^'- 

 labeled thyroxine and shows that the principal excretion product was not 

 thyroxine but an unidentified P^^ component, which was called com- 

 pound U. Purified samples of compound U were obtained by paper- 

 chromatographic separation from other P^^ components of bile. A rat 

 was injected with P^^-labeled thyroxine, and the bile collected between 

 the eighth and fourteenth hour postinjection. The bile was brought to 

 pH 3.5 to 4.0 with dilute HCl and extracted with butanol, and a total of 

 200 lA of the extract delivered in 25-^1 portions to each of 24 filter-paper 

 strips. Compound U was separated by chromatography as previously 

 described, and the sections of paper corresponding to compound U were 

 cut out and eluted with dilute NaOH. Figure 8-8, right, demonstrates 

 the purity of the compound U preparation thus obtained. The eluates 

 from the 24 strips were combined for administration to other rats for 

 study of the metabolism of this particular substance. 



An illustration of quantitative determination of labeled bromine 



