The Use of lc Labeled Tyrosine to Explore the Synthesis of Thyroid Hormone The 131r studies dealing with the synthesis of iodinated led to additional work attempting to use a second isotope. Same unds in Fabbits J4c labeleli tyrosine with the label in the carboxyl position was incubated with thyroid slices ih a nutrient solution to study the sequence of the synthesis of thyroid hormone. As expected, the l'tc labeled tyrosine was recovered in its original form in the thyroid tisaghe slices but none of the l4c was found in the mono- or diiodotyrosine fractions. It[was surprising then that some of the 1c appeared in the thyroxine and triiodothyronine fractions recovered from these slices. Because of the longstanding and unipersally attractive theory that the synthesis of thyroxine is accomplished by the combination of two icdotyrosine molecules, this apparent by-passing of mono- and diicdotyrosine to form thyroxine was unexpected. See attached Figure 3. It should be poifhted out that the yields of l4c in the thyroxine and triiodothyronine fractions were very smll but Significant. This was reported in the discussion of the paper by Gross andj Pitt-Rivers at the Laurentian Hormone Conference in September, 1953. Dobyns, B.M. In discussion of a paper by J. Gross and R. Pitt-Rivers. bid Hormone Physiology and Biochemistry. Triiodothyronine in Relation to Thyroid Physiblogy. Recent Progress in Hormone Research 10: 119-121 Academic Press, 1954. Although the experiment demonstrating the phenomenon of tyrosine by-pabsing of the mono-— and diicdotyrosine to form thyroxine had been successfully repeated before the observation was reported, it was looked upon by others with same skepticism} The experiment has since been performed a total of 7 times confirming the synthbsis of a small amount of thyroxine from tyrosine without forming mono- or diiocdctyropine fram the labeled tyrosine. These incubation experiments have been performed usilhg i3ly to label iodide and l4c +6 label the tyrosine in the same incubation experiments. ‘The two isotopes were counted in the various fractionated samples and their distribution determined by first counting the 15ly and then permitting and then counting the l4¢, ‘see attached Fi 4. The all of this isotope to decay 131I iodine was_abupdant in the mono- and diiodotyrosine but there was no 1%¢ in these compournis. The l4c sine again appeared in the thyroxine and triiodothyronine fractions in small but] significant amounts . Paper Chromatography of Iodinated Campounds Although it was possible to completely separate the iodinated compoundg by the starch column method developed and described, it was so laborious that it rpquired almost a week to run a single separation and count all of the samples from ft. It was therefore_impossible to use this method of separation for a series of simples. containing 131; and collected over a period of hours or a few days because 2 small. amount of isotope in iodinated campounds decayed before it could be measurefi accurately. During and following the development of the starch colum chromatography, paper chromatography gained increasing popularity. Our method of paper chromatogrep was developed as a quantitative method using a butanol-dioxane—ammonia solvpnt descending fashion. in a2 This we developed for our own use from one of the two Bteps of the two dimensional paper chromatography originally described by LeBlond and Grpss. general pattern became very popular and was widely used. The paper method using butanol as a principal ingredient of the solvenk This in an acid medium was extensively studied in our laboratory because it was rapid Bnd seemed reasonably quantitative. Much time was spent studying this method as it wak being used by others. In the light of our experience with the acid medium used originally