,1 .fi. 'fl-tg’t R If ; v'€".""' fit» :1 ‘9" fl" ’.'-.4 ”on." saw“; gag..:....\.,3. 1w. ~.,v -< .» . - n o ~ :1- 9"Qv - .c “‘3-0 I: n" :- ?CP“ ,m- 0“ v r ‘-i' ‘ I; ' * ‘50—! ’\3 ‘f r‘.$rifr“-Z m'} :v‘ (A t' ‘!:v 1 «5‘ m 51 ' ’H‘ _ ‘v‘.‘. j é'x‘uN‘I‘xr . cm" {.r "1" . m.“ we: . . x: = '1 .. ... -..-'. - I‘m q‘ a: w-u .0... 3'." 'W; “n 1 z ', ‘Cphn't 9, (ffV? ' ".Q.'”"ia\ I A 3‘“. c = R if?! "" '9 "3'4": ‘ ‘ ' '. '3. n- v v: ,o r, ,- _-: a .s f '. . 1'2 ‘4 «’1‘ 3“" ‘3!» . {Q ’m K. {1 n ', ‘1. u T'," ‘2 ,.£(I '. o: ' I’fi.’ _ 2 I}, J: n '9‘ I“‘ ; “ . 0' . u L I' ‘W' t (u ‘1" J u 0 5 . ; nu». I . - .0 a :2'1’ O {f a .I'O' ’ p . q C‘ 0, fry-4t ‘90 u ,‘,‘. we; . o ‘3. “In: ‘5’. . % mkt'g-h‘ \. :‘c ‘3‘ ' -"r'-'::r’:- :7 W! h: .«Rm- : m: J.'_ . . 79. . . . 1. . ‘ 'v-.‘. F!“- Ikuutt 134.131".€'s u-.1'\' . u. \z'w: m+ucsfk .0! ‘ I. r-v; .- I Q. n \a'akhk “1' !.' 9 r“ -.- 1'". t-‘I \\ it; x ‘ This it to certify that the l thesis entitled A Critical Analysis of the Butyl .j Alcohol Extraction Technique Used : in the Quantitative Determination -7 of Thyroxine. presented by t wallace Friedberg has been accepted towards fulfillment of the requirements for -‘ _M_o__§_t_ degree in_2hy_&1.0.logy . :- Major professor Due November 26, 1951 0-169 L.‘ .'_m.. - ._- v.--"' —GJM- A CRITICAI.ATALYSES C? r" T‘flf“ ”x 71'3") '57er {Th 'r L -1t'~.'1‘;),rv-‘-r «f m “fi'f‘”? . . 134.1 f T 1LJOiC .44 I LAVA- ”CA. 141"" W—LLJ ‘5 t.,t..l_.L’ .5; In "‘7'? C.*T.z“"‘Tr“‘"’”9”I w? P‘""‘TI‘TTmTO" C77 'T‘Tr‘fv J‘ -‘.--—-J \‘- AbI-L-L-a-JL J J .JJJi_ .J.‘~L‘ .t. I15“ .L -0 1‘ *e“-— 4‘. A .J Eallace Friedberg Submitted to the School of Graduate Studies of Tichigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of ",~fVm‘-1~w _“‘\ p- ..‘ - not :55. z: SCILCS; Department of Physiology and Pharmacology .‘J‘ ACKN OWLEDG‘ TENT The author wishes to express his appreciation.to Dr. E. P. Reineke of the Department of Physiology and Pharmacology for his guidance and advice throughout the course of these investigations, and during the prepara- tion of this manuscript. He a130'wishes to thank Dr. B. V. Alfredson, head of the Department of Physiology and Pharmacology, for the use of the facilities of the department, Dr. C. D. Hause of the Department of Physics and Astronomy for making available an Ansco-Sweet Densitometer, and Dr. W. S. Lundahl of the Department of Biological Science for instruction in the preparation of radioautograms. many thanks are due Hr. John Mbnroe for caring for the animals used in these experiments. Finally, the author wishes to express his thanks to the Michigan Agricultural Experiment Station for support of the project under which this work was carried out. ********** ******** ****** **** ** * ITITI-ICDITCTI13.0.0...OOIIOOOOOOOOOOOO0.0.0.0000... R37 I 5:"! OF - are LI“? «‘1‘ AiATTmEOOOOOOOOO00.000.000.00. .J Absorption and Utilization of Thyroxine... Optical Activity of Thyroxine............. Iodinated Casein.......................... The Putyl Alcohol Extraction of Thyroxine. £3 jRIFJUTAL PROCEDWT“ <1 EUOOOOOOOOOOOOOOOOOOOOOOOOO Preparation of Radioactive Thyroxine from Casein. Io‘iir‘fltion.000000000000OOOOOOIOOOOOOOODOIOOOI Hydrolysis.................................. Extraction.................................. Chromatographic Analysis......................... Preparation of the Radioautograms....................... Hydroxide—Sodium Carbonate "flash Solution...................... Biological Assay of Diiodothyronine. The Efficiency of the Sodium Results...0....OOOOOIOOOOOOOOOOOOOOO Experiment I.... -a fixperiment II... Experiment III.. DISCTTSSICTT. O O o o o . STIT.EBXRYOOOOOOOOOO LITERATURE CILED. APPEFTDIX. . . . . . . . . 10 ll 16 [\3 cammmmm 0019010303!» 03 C») ()1 <1 42 INTRODUCTICF fiumerous instances are cited in the literature in which the biologi— cal potencies of compounds with a thyroxine moiety are not proportional to that anticipated on the basis of chemical assays. In a situation of this type both the chemical and biological assavs may be unreliable. Previous investigators have demonstrated that insofar as biological thyroxine determinations are concerned, consideration must be given to certain "obstacles" which might prevent the active substance from produc- in“ its maximum response. In this respect oral administration involves possible differences in absorption from.the digestive tract. The possi- bility of preferential destruction of one thyroidally active substance over another also complicates the picture. Once absorbed, the various active preparations are not necessarily utilized to the same degree. Different doses of the same preparation administered by the same route could give disproportionate responses. Furthermore, pure thyroxine exists in two optically active forms which must be considered separately. When thyroxine in protein combination is under observation the possible presence of other compounds with chemical and biological similarities to thyroxine should not be overlooked. The present study deals primarily with a critical analysis of the nmst widely used chemical method for assaying thyroidally active proteins, the butyl alcohol extraction technique. A rGVlOW'Of related aspects of the entire problem has been included in order that the limited findings nmy be properly evaluated in terms of the overall problem. -1- Specifically the review will consider: mu 1. Absorption and Vtilization of thyroxine Optical activity of Thyroxine I‘D . 3. chinatea Casein 4. The 7utyl alcohol Extraction of Thyroxine The butyl alcohol extraction technique for the quantitative deter- mination of thyroxine in protein combination was originally proposed by Leland and Foster (1932) and revised by Plan (1955, 1935), Reineke et al (1943) and others. Since this method depends on a final determination of the iodine content it is presupposad that all of the iodine in the washed extract represents thyroxine. To date no satisfactory proof has been offered that this supposition is correct; in fact considerable evidence is now available to indicate that non-thyroxine iodinated com- pounds occnr in this extract. In the present investigation the reliability of the butyl alcohol extraction technique as a quantitative assay for thyroxine in iodinated casein has been evaluated. The chemical aspects of the problem may be divided into several classifications as follows: 1. Iodination of casein with radioactive iodine to facilitate comparative iodine determinations. 2. Controlled purification of the butyl alcohol extract to provide quantitative information as to the percentage of iodine removed. 3. Paper partition chromatognaphy of the extract to establish the presence of any unreported compounds. 4:. Radioautograms of the chromtograms in order to locate the iodinated compounds and determine relative amounts of iodine in them. In view of a report by Iiird and Trikojus (194.8) that diiodcthyronine, a compound with a structure similar to that of thyroxine, was found in iodinated casein, a biological assaj,r was carried out to establish the extent of its thyroidal activity. Earlier workers, using methods based on different principles than employed in the present investigation, re- ported activities ranging from one—twentieth to one-fortieth that of racemic “thyroxine (Rapport et al (1931) and Gaddum (1927). It is hoped that the results presented in this paper will contribute to a better understanding of some of the complex problems involved in the study of "thyroxine activity". -3- Absorption and Utilization of Thyroxine After developing a biological test for thyroxine and thyroid preparations, Cameron and Carmichael (1921) carried on a series of ex- periments in which they administered thyroid and thyroxine orally to white rats and made comparisons by observing decrease in total body weight as well as relative hypertrophy of liver and spleen. On the basis of equal iodine content in the two preparations they observed that thyroid produced from.two to four times the effect of thyroxine. These authors suggested that the differences in effectiveness between the two preparations might be due to bacterial decomposition of the free thyroxine, the thyroxine in combination with protein being more resistant. In view of the work of Kendall (1915), showing that only a part of the thyroid gland contains material with thyroidal activity and the more recent report of Taurog and Chaikoff (1946a), that only 25 per cent of the iodine is thyroxine iodine, the data of Cameron et a1 (1921) can be reinterpreted to indicate that thyroid "thyroxine" has eight to sixteen times the effect of d,l—thyroxine. It is assumed that these investiga- tors used racemic thyroxine inasmuch as an optically active isomer of thyroxine had not yet been isolated, Harington and Salter (1950). During the same year, Plummer (1921) made similar observations in human beings. He reported that absorption of thyroxine from.the intestin- al tract apparently was erratic, although the physiological reaction following its administration was identical to that obtained with fresh or desiccated thyroid glands. Plummer's conclusion conforms somewhat more to present day concepts than the suggestion of Cameron et al (1921) that bacterial decomposition is responsible for the poor effect of orally administered thyroxine. However, his experimental subjects, human beings, are notoriously poor for carefully controlled experiments. Harington and Salter (1930), working with a peptide product of tryptic digestion of thyroid, containing no iodine compound other than thyroxine, found that oral administration of it caused a pronounced in- crease in.the oxygen consumption of rats, while l-thyroxine similarly administered had practically no effect. According to these investigators, the wide range of solubility of the digestion product compared to the relative insolubility of thyroxine made it highly probable that the ab- sorption of the latter would be insignificant while the peptide would be easily absorbed, thus accounting for its high degree of effectiveness. Quoting experiments by J. H. Means of Boston, who found that the digestion product is fully as effective as an equivalent dose of thyroid (oral administration to patients with subnormal basal metabolic rate), Farington et a1 concluded that the increased activity of thyroid thyroxine, when compared to free thyroxine, is probably due to the linkage and the optical activity of the natural thyroxine (optical activity will be dis- cussed below). Using thiouracil-treated chicks after the method of Dempsey et a1 (1945), Monroe and Turner (1949) found crystalline d,l-thyroxine to be 20 per cent as effective when administered orally as when given by sub- cutaneous injection as the disodium salt. On the other hand, desiccated thyroid was about equally effective perorally as parenterally. In a separate experiment on the d,l-thyroxine equivalents of the feed and the feces, it was calculated that iodinated casein was about 85 per cent absorbed. These experiments show rather conclusively that thyroid is more readily absorbed than crystalline thyroxine. Apparently, thyroid is also absorbed better than iodinated casein. Thompson and co-workers (1955), reporting on fine effects of various compounds when administered orally to myxedema patients, observed that the disodium salt of thyroxine is more effective than the monosodium salt and the latter more effective than pure thyroxine. These authors suggested that solubility is the important factor in absorption, since thyroxine is the least soluble of the three and the disodium.sa1t the most soluble. They also cited the work of Barnes who obtained evidence that thyroxine may be destroyed to the extent of about 11 per cent, in vitro, by the action of pancreatic enzymes. It is significant that Mbnroe and Turner (1949) did not find an appreciable difference between the monosodium.and disodium.salts of thy- roxine when.the crystalline salts were administered orally. The apparent discrepancy between the two reports might, however, be accounted for in that Thompson and co-workers administered the disodium salt in alkaline solution. There also may be species differences. -6... Administering an alkaline solution of thyroxine (disodium salt) directly into an isolated intestinal loop, Schittenhelm and Eisler (1932) found that 90 per cent of the thyroxine is absorbed. Harington and Salter (1930) reported their tryptic digestion product of thyroid, containing only thyroxine iodine in peptide linkage, to be slightly more active (oxygen consumption of rats) than an equivalent dose of l-thyrozine when both were injected subcutaneously. In the case of thyroxine in peptide linkage, as it is believed to be in the thyroid gland, it seemed quite likely to them that the active principle is liberated slowly, and therefore is utilized more efficiently. They also suggested that thyroxine, when injected in large doses, is partially lost by excretion before it is utilized by the organism. These considerations should be taken into account when physiological rather than pharmacological properties of the hormone are being studied. The best assays are conducted with small doses over a period of several weeks. More recently, Frieden and Winsler (1948), using the goiter preven- tion method (Dempsey, 1943), reported results which indicate that the biological activity of thyroxine, combined as thyroid protein, is about four times as great as d,l-thyroxine. These investigators determined the "thyroxine" content of the thyroid material by the chemical nethods of Reineke et al (1943) and Blau (1935). Even assundng the dentrorota- tory isomer of thyroxine to have no activity, the thyroid material was found to be twice as active as l—thyroxine. -7- Feng (1950), comparing the calorigenic effects of intraperitoneally injected thyroid and d,l-thyroxine also found thyroid to be more active when comparisons were made on the basis of their l-thyroxine content. The thyroxine iodine of the thyroid was estimated as being 25 per cent of the total iodine (Taurog and Chaikoff, 1946a) and one-half of the d,l- thyroxine was assumed to be biologically active (Reineke and Turner, 1945). Apparently, thyroxine when administered as a protein moiety is enhanced in activity. An alternate conclusion would be that other constituents with thyroidal activity and low iodine content are present in thyroid and iodinated casein. Optical Activity of Thyroxine Foster, Palmer and Leland (1936) found that thyroxine administered parenterally as the pure l-thyroxine or given orally in combination as thyroid was equivalent insofar as its calorigenic effect on guinea pigs was concerned. This was in accord with the finding of Harington and Salter (1930) that thyroxine, isolated by enzymatic hydrolysis of the thyroid gland, is in the form of its lovorotatory isomer. Palmer et al (1935) reported that in guinea pigs, thyroxine admin- istered orally in the form.of thyroid gland produced twice the calori- genic effect of d,l-thyroxine. Apparently therefore, the dextrorota- tory isomer has no measurable activity under the conditions of flqese experiments. In more direct comparisons, Reineke and Turner (1943) found the activity of l-thyroxine, isolated from.iodinated casein, to be approxi- mately twice that of an equivalent amount of d,l-thyroxine, also derived from casein. These investigators used per cent increase of the basal metabolic rate of guinea pigs as a criterion of activity. Again in 1945 the same investigators obtained similar results using other assay methods: l-, and d,1-thyroxine were compared in their ability to reduce the thy— roid weight of thiouracil-treated-chicks and rats and to stimulate meta- morphosis in.tadpoles. 0n the other hand Gaddum.(1929), measuring the oxygen consumption of rats, found that the dextrorotatory isomer possessed a small amount of activity; the numerical ratio varying between 1.5 and 3. When he tested the l-thyroxine on tadpoles by an earlier method (Gaddum, 1927) it was similarly more effective, but the ratio varied between 1.2 and 2. The possibility exists that the activity reported for d-thyroxine by Gaddum.was due to incomplete resolution of the sample. Salter et a1 (1935) when comparing'the calorigenic effect of d-, and l-thyroxine in patients with spontaneous myxedema found the two forms to have essentially identical potencies. Pitt-Rivers and Lerman (1948) also reported that d-thyroxine possessed physiological activity when tested in myxedema patients, but only one-eighth to one-tenth that of l-thyroxine. Observing the effect on the basophils of the pituitary gland in thiouracil-treated rats Griesbach and c0dworkers (1949) reported d-thyroxine to have 0.3 the activity of l-thyroxine; both by direct comp parison of the dextro- and levorotatory isomers and by calculating the results of an assay of d,l-thyroxine. These investigators believe their method to be more sensitive than the thyroid weight or calorigenic techniques. -9- The question of d-thyroxine activity is not closed. However, in most practical considerations no significant error will result if the dextrorotatory isomer is assumed to be without activity. Iodinated Casein ‘Hith the conditions for preparing iodinated casein with high bio- logical activity established (Reineke et a1 1943), it was desirable to know the relative activity of this material. Reineke and co-workers (1945) reported close agreement between bio- logical and chemical assays of iodinated casein, when the apparent thy- roxine content of the iodinated protein was determined by their method (Reineke et a1 1943). Frieden and'Winzler (1948) comparing the parenteral thyroidal activity of natural and synthetic thyroproteins by the goiter prevention method (Dempsey, 1943), reported synthetic thyroprotein to have less activity than expected from its apparent l-thyroxine content. The chemi- cal assay technique of Reineke et a1 (1945) as well as the method of Blau (1935) were used to determine apparent thyroxine. Two normal sodium hydroxide was used as the hydrolytic agent in the latter method (no significant difference was found between the two methods). In 1949, Reineke and cedworkers reported an isotope dilution tech- nique for the determination of thyroxine. Applying it to iodinated casein, they reported thyroxine values which were about 25 per cent of those obtained b7 the butyl alcohol extraction method of Reineke et a1 (1943). -10- Uhen the data of Zrieden and‘Xinzler (1948) are corrected for true V thyroxine, on the basis of these findinbs, tne biological activity is found to be almost twice that expected from the chemical assay. Feng (1950) found no significant difference between the calorimenic effects of thyroid and iodinated casein when preparations of equal thy- roxine content were administered to rats. The thyroxine content of iodinated casein was determined by isotope dilution while 25 per cent of the total thyroid iodine was assumed to be thyroxine iodine (Taurog and Chaikoff, 1946). In 194 Kird and Trikojus established, by chromatographic analysis, the presence of two compounds besides thyroxine, with similar solubility characteristics, in a butyl alcohol extract of the hydrolysate of iodinated casein. One was show,.to be diiodothyronine by its similarity to a reference compound and the other w s believed to be triiodothyronine because of its position on the chromatogram, between thyroxine and di- iodothyronine. This information helps explain the high chemical assay reported by Frieden, et al (1948) for iodinated protein. In other words The Butyl Alcohol Extraction of TherXLne The foundation for thyroxine determinations, based on a quantitative isolation of the compound, was laid by E. C. Kendall. We u s the first to demonstrate the application of alkali for the hydrolysis of thyroid protein as an essential step in concentrating the iodine~containing con- 3 reacing the thyroid protein with IaOH and subsequent U) (I’- 'Jo k 5.5. 9 'd‘ U) 0 ‘ J 1% rt (3 "5 d" -11.. dialysis of the hydrolysate, Kendall (1915) separated the products into two main groups: one containing 60 per cent of the iodine and 9 per cent of the nitrogen and the other 40 per cent of the iodine and 91 per cent of the nitrogen. In 1915 he reported that upon hydrolysis in alcoholic NaCH, desiccated thyroid was altered such that the iodine was in two forms of organic combination. Approximately half of the total iodine was soluble in acid while the other half was acid—insoluble. Furthermore, the acid-insoluble fraction was capable of producing the symptoms of hyperthyroidism as well as relieving the symptoms of hypo- thyroidism. ‘With these two fundamental principles firmly established: (1) hydrolysis on the thyroid protein and (2) association of the physio- logically active material with the acid-insoluble portion of the hydroly- sate, Kendall continued his investigations and succeeded in isolating the physiologically active compound in a crystalline state (1915, 1919). Iarington and Randall (1929) devised a chemical assay for thyroxine at the request of the British Pharmacopea. Thyroid protein was hydro- lyzed by boiling it in.£ HaCH solution for four hours. The filtrate was adjusted to pH 5 with 50 per cent 92304 and the acid-insoluble iodine was considered thyroxine iodine. Leland and Foster (1952) contended that Harington and Randall's analysis for thyroxine, based on its insolubility in acid, although ex- tremely simple technically, did not completely excbzde diiodotyrosine from the "thyroxine" fraction. Their conclusion.was based on an experi- ment in.which they rehydrolyzed the acid-insoluble fraction, prepared by the previous method, in 2? HaCH for 18 hours, extracted it with normal butyl alcohol and washed the alcoholic extract with Z HaCH. They ob- served a strong nitrous acid test on the wash solution which was assumed to be due to a large diiodotyrosine fraction. An.analysis for inorganic iodide, according to the method of Foster and Gutman (1930), indicated only a small amount and convinced them that there was no serious des- truction of thyroxine. Furthermore, on comparing per cent thyroxine iodine by their butanol extraction technique with the method of Harington, et a1 (1929) on the same sample of desiccated thyroid, Leland and Foster found the former technique to indicate a thyroxine content about half that determined by the acid-insoluble iodine analysis. In their method for thyroxine determination the latter authors hydrolyzed desiccated thyroid for 18 hours with 2? UaCH, extracted the aqueous mixture with normal butyl alcohol and washed the alcoholic extract with E HaOH to re- move the remaining diiodotvrosine. The hydrolysis with E NGOH was recommended to increase the yield of thyroxine. As judged by recovery experiments, unavoidable destruction of thyroxine during alkaline hydrolysis was about 15 per cent. Blau (1935) modified the extraction procedure of Leland, et a1 by carrying out the initial step in an acid medium. The thyroxine values obtained by this modification were about 11 per cent higher than by the former method. He also introduced an alkaline washing solution which afforded a more desirable distribution.between the thyroxine and non- thyroxine iodine fractions. In 1935 Blau reported that a shorter period of hydrolysis was neces- sary when Ba(-H)2°BH20 was the hydrolytic agent. In recovery experiments, -13- comparing the stability of thyroxine when boiled for six hours in eight per cent Ba(OH)2-8320 and ZE'NaOH, the recovery of the thyroxine iodine was on the average six per cent higher by the former method. Blau's technique for quantitative thyroxine isolation consisted in hydrolyzing desiccated thyroid (or fresh thyroid) in eight per cent Ba(OH)2.8H20 solution for six hours, adjusting the pH of the hydrolysate to about 3.7 with HCl and extracting with normal butyl alcohol. He then.washed the extract with a solution consisting of 4§_HaCH and five per cent Eazcos. The recoveries of thyroxine from.mixtures of thyroxine and di- iodotyrosine dissolved in an eight per cent Ba(OH)2°8H20 hydrolysate of testicular powder averaged close to 100 per cent and the effect of in- organic iodine was invariably too small to measure. Comparing the apparent thyroxine content of iodinated casein by the method of Blau and by a tadpole assay technique, Reineke and co- workers (1945) found that results by the former method were considerably higher than the latter biological assay. This led them to the supposition that the high chemical assay might be due to incomplete hydrolysis of the protein, with the result that some non-thyroxine compounds were soluble in normal butyl alcohol. In comparison with the former method, the re- sults obtained by a 20 hour hydrolysis with 40 per cent Ba(OE)208H20' showed far better agreement with the biological assay. In view of the fact that the apparent thyroxine content did not change significantly between periods of hydrolysis from eight to twenty-eight hours, 20 hours was recommended. -14... Investigatinr the thyroid content of rat thyroid glands, Taurog and Chaikoff (1946) found that values obtained with Ba(OE)2-9320, using HaOH as the hydrolytic agent. However, they did confirm Blau's finding that NaOH caused greater destruction of thyroxine than Ba(OH)2°8320. It should be pointed out that Blau (1935) also reported his technique to give variatle results. Recent reports, reviewed in other sections of this paper, suggest that the butanol extraction technique, even under the most accepted con- ditions, is not specific for thyroxine. -15.. 21x 3:“ arm; PT1C~CEDTTIIE Preparation of Radioactive Thyroxine from Casein The method described by Reineke and co-workers (1943) was used with only minor modifications. Iodination Twenty grams of casein (purified, vitamin-free), nine gms. of sodium bicarbonate (C.P.), and 670 ml. of distilled water were mixed in a 29 cm. by 10 cm. glass cylinder. Through a rubber stopper, fitted snugly into the mouth of the cylinder, a short length of metal tubing was inserted to permit the passage of an electrically operated glass stirring rod, bent into a triangle at the end to facilitate stirring. Radioactive iodine-131 solution.was prepared from a radioactive iodide solution in a 50 ml. test tube, by the addition of a small crystal of potassium iodate and one drop of concentrated phosphoric acid to about 1 ml. of the iodide.* A small sodium iodide crystal was added and pro- duced a color change, indicating that iodine had been released from the iodide. Five ml. of distilled water were then added to the solution. The glass cylinder was placed in.a constant temperature thermostatic- ally controlled water bath and the heating unit and stirring apparatus were turned on, the latter being set for rapid stirring. When the temperature of the bath had reached 40 degrees centigrade, stirring was (0 topped and the radioactive iodine solution, previously prepared, was Purchased from Oak Ridge Yational Laboratory, Carbide and Carbon Chemicals 00., Oak Ridge, Tenn., and used in concentrations of approximately 10 mc. per m1. w) -1 J- added to the mixture in the cylinder. The tube was rinsed with five ml. of water which was also added to the mixture. Stirring was resumed for several minutes during which time 3.6 gms. of iodine crystals Were 'weighed out on a filter paper. Stirring was interrupted while the iodine crystals were added to the mixture in the cylinder, and then re- sumed and continued for 18 hours at a temperature of 70 degrees centi- grade. Hydrolysis After the 18 hour incubation period the cylinder was removed from the water bath and allowed to cool at room temperature for about one- half hour. Sufficient 3.52-HC1 was added to precipitate the protein, and caprylic alcohol was added dropwise to reduce foaming. 'fhe mixture was then filtered through a Buchner funnel by means of negative pressure. The protein in the funnel was washed by adding .52.HC1 in sufficient quantity to cover the precipitate. flashing was enhanced by gentle stir- ring and the liquid was rembved by suction. The precipitate was washed twice in this manner after which the damp precipitate was put into a 500 ml. reaction flask and 129 rd. of distilled water, 64 gms. of Ba(OH)2°BH20 and about one-half ml. of caprylic alcohol were added. The mixture was boiled gently under a Liebig reflux condenser for 20 hours, the flask being agitated frequently by shaking until it was apparent that there was no further danger of foaming. Extraction After the 20 hour hydrolysis, heating was discontinued and the hydrolysate was permitted to cool until the barium salts had settled and the flask could be handled easily. The supernatant liquid was de- canted and saved. A mixture of g_butyl alcohol and 3.5 E_HC1 was added to the barium salts in amounts of approximately two and five ml. re- spectively. The barium salts were then vigorously stirred in this butyl alcohol-acid mixture to aid in their solution and suspension. The mix- ture of barium salts was combined with the supernatant liquid and 5.5'E HCl was carefully added to the mixture, from a burette, until it was acid to congo red paper. Approximately 40 ml. of the mixture were re- moved to a separatory funnel for extraction of "thyroxine", and about 20 ml. of E_butyl alcohol were added. The aqueous and organic phases were mixed by swirling the mixture around in the funnel. After the two phases had separated the butyl alcohol (upper) layer was removed and washed with an equal amount of a solution prepared by dissolving 480 gms. of NaCH and 150 gms. of NaZCO3 in water and making up to a volume of 5000 ml. Separation of the wash solution and the butyl alcohol extract was per- mitted to take place over a period of not less than one hour. The wash- ing and separation procedure was repeated with one-half the volume of wash solution used the first time. Chromatographic Analysis The "ascending" technique for chromatographic analysis,developed by 'fiilliams and Kirby (1048), was adapted to the study of the characteris- tics of the purified butyl alcohol extract of the hydrolysate of iodi- nated casein. Solvent mixtures used by Hird and Trikojus (1948), in a study of thyroxine and its analogues, were also found to be satisfactory for the purposes of this experiment. The apparatus consisted of a 43.5 cm. by 20 cm. glass cylinder in the bottom of which was placed a nine cm. Petri dish. The cylinder was fitted with a plate glass cover and the seal was made air-tight with desiccator grease. A 40 cm. by 28 cm. sheet of Whatman No. 1 filter paper was marked udth a pencil line parallel to the shortest axis and three cm. from the end of the sheet. The filter paper was rolled into a tube and the edges were stapled together. Solutions of the samples to be analyzed were placed in spots at intervals along the pencil line en- circling the bottom. For purposes of comparison the paper was also Spotted with thyroxine, diiodothyronine, and diiodotyrosine. A dropping pipette,'the tapered end of which had been heated in a flame until the lumen was reduced in size, was originally used to apply the solution, in later studies the flow of liquid was found to be more easily controlled with a 0.5 ml. pipette. Small spots were found to give the most repro- ducible results. After being spotted, the paper was dried at room temperature for at least 15 minutes. The solvent mixtures were prepared by shaking together in a separatory funnel, a mixture of 120 ml. of E amyl alcohol, 120 ml. of E butyl alcohol and 240 ml. of 23 NH40H.* The organic and aqueous phases were then allowed to separate for a period of time not less than one hour. The aqueous (lower) layer was drawn off and an amount was placed in the glass cylinder just sufficient to cover the bottom. The Petri dish was placed in the cen- ter of the cylinder and the paper tube was placed, penciled end down, in mm * In the earlier experiments, in which the radioautograms were pre- pared, the solvents did not contain amyl alcohol. Instead, twice the amount of butyl alcohol was used. -19- the dry Petri dish. The cylinda‘ was sealed with the plate glass and the tube was allowed to stand in the Petri dish for approximately three hours to enable the paper to become impregnated with the queous phase. The paper tube was then removed from the cylinder and 32 ml. of the organic phase were dispensed from the separatorv funnel into the Petri dish. The tube was again placed in the same manner as before and the cylinder was sealed with the plate glass. The solvent front was allowed to move up the paper for varying Ho per ods of time, up to 30 hours. In all cases, the paper tube was re- mover~ from the cylinder before the solvent had reached the top. The paper was flien dried at room.temperature for several hours. By means of an atomizer attached to an airline, the dried paper was sprayed, until saturated, with a 0.4 per cent solution of triketohydrindene hydrate (ninhydrin) in E butyl alcohol, in order to render the amino acids vis- ible. The soaked paper was then allowed to stand in.a drying oven at about 75 degrees centigrade for approximately 15 minutes. Blue spots of various shades appeared, representing the compounds in the mixture. The Rf values were determined by dividing the distance the front of the spot in question moved by the distance the so v at front moved, the ’origin in both cases being the point of application of the spot. Preparation of the Radioautograms Radioautograms were prepared by placing Kodak To Screed I—ray film in direct contact with the chromatogram, between two flat pieces of glass. The procedure was carried out in complete darkness. Exposure ‘1 , I ligure 1 Apparatus for Paper Partition Chromatography E‘I GPRE l '- I, \_ ""“") FIGJ APPAflATUS F00 AMER PAR TIT CHROMATOGRAPHY ION time was determined by the Beta count per minute, of the portion of the chromatogram to be recorded. An unshielded mica window Geiger Mueller tube was placed directly over the chromatogram. A 24 hour exposure was sufficient for a Reta count of 5000 per minute. The autogram.in Figure 2 was exposed for six days. The exposed autograms were developed and fixed with Kodak X-ray developer and Hypo, at full strength. Densitometer readings of various areas on the developed radioauto- grams were taken by means of an Anseo-Sweet Densitometer. The Efficiency of the Sodium.Hydroxide—Sodium Carbonate wash Solution Radio activity determinations were made of samples of the butanol extract of the acid-insoluble portion of the hydrolysate of iodinated protein with a scaling unit in conjunction with a Geiger Mueller tube. The samples were taken from; unwashed extract, extract washed once, and twice-washed extract. The wash solution consisted of a dfi'NaOH and five per cent Nazco3 mixture as originally described by Blau (1935). An amount of wash solution equal to the volume of butyl alcohol extract was used in the first washing while one-half of that volume was used for the second washing. The samples were evenly distributed on an aluminum disc and dried at room temperature before radioactivity determinations were made. Biological Assay of Diiodothyronine The thiouracil technique described by Dempsey and Astwood (1943) was used to assay purified diiodothyronine. Groups of six male albino rats purchased from.Rockland Farms, New'York, were used throughout the experiment. The rats were fed ad libitum with a standard diet devised by Dr. C. A. Hoppert, Department of Chemistry, Michigan State College, (see appendix) and drinking water containing 0.2 per cent thiouracil was available at all times. The thiouracil was put into the drinking water one day before the injectionS'were begun, and for 14 days the rats were given daily subcutaneous injections of the appropriate amount of d,l-thyroxine or d,1-diiodothyronine. 0n the tenth day one rat in group twO'was found dead. The test compounds were dissolved in 0.1g NaOH and the pH was adjusted to about eight with dilute HCl. Six rats were kept in each cage and the room temperature was 24jd degrees centi- grade. Artificial lights were turned on from approximately 8:00 A.M.'to 5:00 P.M. After the 14 day experimental period all the rats were sacrificed and the thyroid glands were dissected out and trimmed free of fat. The rats were weighed to 0.1 gm. on a triple beam balance and the thyroid glands to 0.1 mg. on a Roller Smith balance. The thyroxine was supplied by Dr. E. P. Reineke of the Department of Physiology and Pharmacology and the diiodothyronine was purchased from S. A. F. Hoffmann—La Roche and Company, Basel, Switzerland, and further purified by the author (see appendix). Results Experiment I Inasmuch as recent evidence indicates that several non-thyroxine compounds are carried along with thyroxine when iodinated casein is assayed by the butyl alcohol extraction technique, it was of interest to determine the number of these compounds. Paper partition chromatography was utilized to separate the com~ ponents of the twice-washed butyl alcohol extract of iodinated casein. The rsults indicate the presence of three compounds other than thyroxine, triiodothyronine, and diiodothyronine (these last three compounds were reported to be in an unpurified butyl alcohol extract, by Hird and Trikojus, 1948*. An intense blue spot appeared when compound III, Rf 0.20, was treated with Ninhydrin reagent (an amino acid indicator). Compound I, Rf 0.03, and compound TI, Rf 0.10, gave comparatively weak ninhydrin tests. Control runs with d,l-thyroxine indicated an average Rf of 0.41. 'Individual results are listed in table I. Experimeni TI Since the butyl alcohol extraction technique depends on an iodine determination, the non-thyroxine iodinated compounds on the chromatogram were located and their concentrations, in a relative manner, were indi- cated by the preparation of radioautograms. An autogram prepared from a chromatogrammed extract, after purification with a sodium hydroxide- sodium carbonate solution,* revealed a spot, 7, just below a thyroxine contro1. Although the control, RP 0.53, was abnormally high when com- pared to the average of thyroxine controls (0.41) run with only slightly different solvents and listed in Table I, the spot in question bears the * In this particular experiment the butyl alcohol extract was washed twice with equal amounts of each solution. -24- Same relation to the thyroxine as the intense blue spot Rf 0.20, de- scribed as compound III in the results of the preceding experiment, i.e., it would seem that spot 7 is correspondingly high. An examination of the radioautogram prepared from unwashed butyl alcohol extract re- veals six spots below spot 7. Densitometer readings of radioautograms made from chromatogrammed extracts (Table II), before and after washing, indicated that the sodium hydroxide-sodium carbonate wash solution has a specific action in remov- ing compounds below spot 7. Readings at 7 were 0.61 for the unwashed and 0.37 for the washed extracts. Moving up the autogram.from.spot 7, readings taken at points directly opposite one another had values of 1.49 and 1.49, 0.92 and 0.82, 0.63 and 0.62 for the unwashed and washed extracts respectively. A.visual inspection and densitometer readings of the area below spot 7 indicated almost a complete removal of iodinated com— pounds by the wash solution. Experiment III Radioactivity measurements of aliquot samples of a butyl alcohol extract were carried out in order to determine the actual change in iodine Concentration before and after purifying with the previously described wash solution. This would give some indication of the effi— ciency of this part of the butyl alcohol extraction technique. Results are recorded in Table III. The average count of an aliquot before puri- fication was 16.2 and after the first and second washings it was 7.9 and 7.2 respectively. The per cent change in counts was 52 after the first washing and 8.4 after washing a second time. -25- TABLE I Rf quums 0P3 POTs PILCT'T“”>0*I:3 (AS'T‘UI"T) 0h '..'T”C' cits P: IPAP‘DP :0T TT3 PTTIPI 30 P‘I'DI LISATE 0P IODI“TTE CIS3IT. THE SPCT S T333 "333 VI I313 TITT TNTYDII" T“"3:T. YAC‘”"" TTYPOKIJE cofTPOL'VALmIs ARE IT. CLTDL‘ Preparation Spot Fumber Tayrozcine Yo. I II III Control 1 0.06 0.12 , 0.24 --- 2 --- —-- 0.20 0.40 3a 0.03 0.12 -—- -_- 3b 0.03 "'"""' 0021 004.5 30 0.01 0.08 0.20 --- 3d 0.04 --- 0.18 0.41 36 0.03 0.07 0.20 0.56 3f """" 0010 0020 004]. _26- DEE-7811‘ ,‘IIIEI 33.119: :03 F MDICAIOI: m 'S :::3PA311§D 30': c I‘0‘30001;?" BUTYL ALCOHOL @3303." 0:? first-:3 :II'UIOLYSITE CF T0“I‘“'“‘ 03331:. It, 3:31 3:23 AID mm: -.x ”QWYI‘V‘ ..I3 a. A 5:03:17: :::'*)r._0.;-LIDI—.--,0:)IU:: cm p0“"‘3 SOLTTTI CN Point On Densitometer Reading* Radioautogram 0nwashed Twice flashed 10 0.63 0.62 9 0.92 0.82 8 1.49 1.49 7 0.61 0.37 6 0.52 0.28 5 0.66 0.22 4 1.06 0.22 3 1.0 0.20 2 0.80 0.22 1 1.12 0.28 Background reading 0.06 Densitometer readings are on a log rithmic scale. Thus, readings of l and 2 would indicate 10 per cent and 1 per cent transmission of light respectively. F I CURE 2 Figure 2 Radioautomrams of a chromatocrammed butanol extract of the hydrolysate of iodinated protein, before ani after purifying the extract with a TaCL—TuZCCS solu- tion. TABLE III 23010.1 TITITY DET .JZ"TT’I'"TO‘T’~" I}? COW-“'33 P333: SEC STD m.0 03 ICTD .133 79. PETE: 2 YIITG 17-? I’"D-.CLISA CF ICDITTATED P210? 3117? - "“31 A SODITT‘YI Tr’if’“) CKlDA-‘ICDI C1230" {323 SC'LTICTI. CCUTTS .133 CCEIEC'Tr ‘D1 03 BACKGRCUID. Group No. Unwashed flashed Once Washed Twice I 16.97 7.85 7.10 II 16.41 7.86 7.4% III 100-32 7096 7006 Average 16.20 7.89 7020 Per cent change in radioactivity after 52 8.4 purification Erperiment IV A biological assay of diiodothyronine was carried out inasmuch as_ this compound is believed to be present in flue purified butyl alcohol extract of the hydrolysate of iodinated casein and since it is available in a pure state. Possibly, a biological assay weuld be more specific for thyroxine than the butyl alcohol extraction technique. Results of the assav, tabulated in Table IV, and depicted graphic- ally in Figure 3, indicate a normal thyroxine response with four groups of 1. 2. 3. and 4 ng. doses, respectively, of d,l-thyroxine. On the other hand, groups administered 10, 15, 20, and 25 meg. doses of d,l- diiodothyronine showed a wide range of activity within each group and none of the doses administered to thiouracil-treated rats showed any significantly greater ability to reduce the thyroid weight than a 1 mcg. dose of d,l-thyroxine; at the 10 and 25 meg. levels of diiodothyronine there was very little, if any, reduction of thyroid'weiqht. At the 15 and ZOnump levels of diiodothvronine the average thyroid weights were very similar and about the same as those in the group administered 1 mcg. of d,l-thyroxine. -30- TIYRCID HEIGHTS PER 100 633. KAT WEIGHT 0? THIOVRACIL-THEATED aAiS AfliafiIS 333’ THYRCKITE AID DIICDOTHIROTITE d,1-thyroxine Group I II III 1? ‘Dose 1 meg. 2 meg. 3 meg. 4 meg. 12.47 11.13 12.26 10.34* 10.42 12.77 11.78 19.04 15.48 10.62 8.2 8.17 14.71 14.65 10.20 9.84 15.31 9.20 12.60 7.32 13.86 8.89 9.40 10.50 Averag 13.70 11.21 10.73 9.23 d,l-diiodothyronine Group I II III IV Dose 10 mer. 15 meg. 20 meg. ._?5 meg. 19.2 18.47 11.14 19.22 33.88 12.28 10.98 8.43 27.61 16.01 13.12 20.12 21.96 11.75 15.40 16.37 15.93 9.85 13.12 19.25 16.25 18.27 14.59 Average 22.47 13.67 13.67 17.99 Control (0.2 per cent thiouracil) 14.68 17.61 10.2 11.98 17.15 12.06 Average 13.95 4: Excluded from average. '0 7‘ or? ‘a .3. U x—IuaJ 3igure 3 D The relation between dosage and thyroid weifiht per 103 EYE. body weight of thiouracil-treatei rats administeroi 1;— . ‘l gcaobdyronine and thyroxine. THVROID WT. PER/00 GM. BODY ERCR \ Q 0105wa - - 7' H/OURA C /L DI/OD 0 TH VRON/NE 7' H VROX //VE L/ _ ._ l l l / 2 J 4 D.L-7HVROX/NE. TBA/LY /0 / 5 20 25 0.1 -D//ODOTHYRON/NE. Y DA /L V DISCUSSION A study of the washing technique, originally described by Blau (1935) for the purification of the butanol extract of iodinated casein hydrolysate, has more completely defined its action in removing non- thyroxine compounds. Radioautograms of chromatogrammed butanol extract, before and after purification, have revealed that sodium hydroxide-sodium. carbonate wash solution specifically removes all but traces of butanol- soluble iodinated compounds with Rf* values below that of spot 7 (approx- imate Rf 0.20); the compound represented by spot 7 is only partially removed by the wash solution. Three distinct spots, presumably repre- senting at least three different iodinated compounds, have been observed which were not significantly affected by the purification procedure. Inasmuch as the concentration of thyroxine dissolved in acid butanol is known to be unaffected by a sodium.hydroxide—sodium carbonate wash solu- tion (Dlau, 1935), it is presumed to be one of the 3 spots. A thyroxine control run adjacent to the unknown mixture suggests that spot 8 repre- sents thyroxine. The other compounds with higher Rf's have solubility properties similar to thyroxine, at least with respect to the particular solutions used in the experiment. These findings indicate that the main sources of error in the standard technique for determining thyroxine in iodinated casein are iodinated compounds similar in solubility to thyroxine. It seems possible that a similar situation exists with respect to thyroid gland. Hird and * . 1 Ratio between rate of movement of compound and rate of movement of solvent front. -33- Trikofius (1949) first reported the presence of amino acids in the butyl alcohol extract of iodinated casein hydrolysate which they tentatively identified as thyroxine, triiodothyronine, and diiodothyronine. Their conclusions were based on Rf values and ninhydrin tests. However, their work gives no indication of the number of compounds in the butyl alcohol extract after the purinication procedure sugrested by “lau. Furthermore, their report offers no direct evidence of the presence of iodinated compounds. The finding of Frieden and Winzler (1949.) that synthetic thyropro- tein showed less thyroidal activity than expected from.its apparent l-thyroxine content can now be explained by he presence of several thyroxine-related but relatively inert iodinated compounds. Support is given to the reliability of the isotope dilution tech- nique used by Reineke and co—werkers (1949) for thyroxine determinations. Their technique gives thyroxine values which are much lower than those obtained by the butyl alcohol extraction method. Preliminary results of direct radioactivity determinations of spots on paper chromatograms reveals thyroxine-iodine activity to occupy only a small percentage of the total activity. The data presented in this paper indicates that the slowly movin compounds, including diioddflmmonine, are removed in the purification pro- cedure. The Rf of diiodotyrosine is 0.00-0.01 (hird and Trikojus, 1948). This is not in accord with the views of Borrows and co-workers (1949). These workers made their analysis polaroqraphieally and reported con- siderable amounts of diiodotyrosine in the purified extract. In their wash solution they used sodium bicarbonate whereas 81au's method calls -34- for sodium carbonate. This could be a misprint or, if not, may account for the apparent discrepancy between the two sets of data. The chromatograms and radioautograms indicate the presence in the twice-washed butyl alcohol extract of a hitherto unreported iodinated amino acid with an Hf of 0.20. The amount of blackening of the X-ray plate used in preparina the autograms and the intensity of the ninhydrin test reveals the compound to be present in more than trace amounts. It is concluded from the number of spots on a radioautogram that six compounds, present in the unwashed extract, have been greatly re- duced in concentration by the purification procedure. A two dimen- sional chromatogram of the unwashed hydrolysate, prepared as a prelimi- nary experiment to a future investigation, indicated the presence of more than 11 compounds giving a positive ninhydrin test. Further evidence of the specificity of the sodium.hydroxide-sodium carbonate wash solution was supplied by a controlled washing experiment; radioactivity measurements indicated that 52 per cent of the iodine was removed by the first washing while a second'washing succeeded in remov~ ing only eight per cent more. A preliminary study of the biological action of diiodothyronine, a compound in the butyl alcohol extract (Hird and Trikojus, 1948) with a structure similar to thyroxine, suggests that it has a qualitatively different action on the pituitary gland; the administration of progres- sively increasing doses, exceeding those reported to have an effect on the metabolism of rats (Gaddum, 1929), did not show proportional effects, if any at all, in reducing the thyroid weight of thiouracil-treated rats. -35.. It is not unlikely that thyroxine is the only compound derived from iodinated casein with its unique effect on the thyroid-pituitary axis; increased thyroxine administration causes a decreased secretion of thyrotropic hormone by the pituitary gland (Aron et a1, 1931). Formerly it was believed, on the basis of metabolism assays, that there was only a quantitative difference between thyroxine and diiodothyronine. Of practical interest is the availability of a biological assay which these preliminary experiments indicate to be specific for thyroxine. -35- (1) /\ to v (3) (4) SIT"? ‘3 TARY Casein was iodinated with a mixture of iodine-127 and radioactive iodine—131. The iodinated protein was hydrolyzed with barium throxide and the acid-insoluble portion of the hvdrol‘sate ex- d .- v tracted w'th normal butyl alcohol. The alcoholic extract was washed twice with a mixture consisting of five per cent “anC 3 and 4N NaCH. Radioactivity measurements indi- cated a 52 per cent reduction in iodine content after the first washing and an eight per cent further reduction after the second 'washing. The purified extract was chromatogrammed and amino acids were lo— cated by treating with ninhydrin reagent. A hitherto unreported amino acid of Rf 0.20 was found. Radioautograms of chromatogrammed,purified and unpurified extracts 'were prepared and densitometer readings were taken of appropriate areas. The girification procedure reduced considerably the concen- tration of compounds with if values below 0.20. An exposed area on the autogram just below a thyroxine control suggested that the amino acid with Rf 0.20 is iodinated. Several iodinated compounds were found in the butyl alcohol extract which were not appreciably altered in concentration by the sodium hydroxide-sodium carbonate wash solu- tion used in the purification procedure. -37... (5) A biological assay of diiodothyronine was carried out on thiouracil- treated rats. Results suggest that this compound has no effect on the thyroid-pituitary axis. ITERATURE CITED Aron, W. Van Canlaert, C., and Stahl, J. 1931. L‘equilibre entre l'hormone prehypophysaire et l'hormone thyroidienne dans le milieu interieur, al'etat normal at a 1'etat pathologique. Compt. rend. Soc. de biol. 107, pp. 64-66. Blau, N. F. 1933. The Determination of Thyroxine In The Thyroid Gland. J. Biol. Chem. 102, pp. 269—278. Blau, N. F. 1935. The Determination of Thyroxine in Thyroid Substance. J. of Biol. Chem. 110, pp. 351-363. Cameron, A. T., and Carmichael, J. 1921. Contributions to the biochemp istry of iodine. IV. The effect of thyroxine on growth in white rats and in rabbits. J. Biol. Chem., 46, pp. 35-52. Dempsey, E. W. and Astwood, E. F. 1943. Determination of the rate of thyroid hormone secretion at various environmental temperatures. Endocrinology 32, pp. 509-518. Fens. L. 1950. M. S. Thesis, School of Graduate Studies, “ichigan State College. foster, G. L., Palmer, W. W., and Leland, J. P. 1936. A comparison of calorigenic potencies of 1-thyroxine, d,1-thyroxine and thyroid gland. ‘Wifli a note on The Thyroxine Content of the Acid-Soluble Fraction of the Peptic Digest of Thyroid Protein. J. Biol. Chem. 115, pp. 467-477. Erieden, E., and Winzler, R. J. 1948. Comparative parenteral thyroxine- like activity of natural and synthetic thyroproteins studied with the goiter prevention method. Endocrinology 43, pp. 40-47. Gaddum, J. I. 1927. Quantitative observations on thyroxine and allied substances 1. The use of tadpoles. J. Physiol. 64, pp. 246-254. Gaddum, J. H. 1929-30. Quantitative observations on thyroxine and allied substances. II Effect on oxygen consumption. J. Physiol. 68, pp. 383-405. Griesbach, W. 2., Kennedy, T. H. and Purves, H. D. 1949. The physio- logical activities of the stereoisomers of thyroxine. Endocrinology 44, pp. 445-4480 Harinrton, C. 2., and Randall, S. S. 1929. Chemical Assay of Thyroid Gland. Quart. J. Pharm. and Pharmacol., 2, pp. 501-506. -39- Harin. ton, C. R., and Salter, W. T. 1930. The Isolation of L. Thyroxine rom the Thyroid Gland by the Action of Proteolytic Enzymes. Bio- chem. J. 24, pp. 456-471. '0. h r Bird, F. J. R., and Trikojus, V. H. 1948. Paper Partition Chromato- graphy with Thyroxine and Analogues. Australian J. Sci., 10, pp. 185-187. Kendall, E. C. 1913. Studies in thyroid activity. I. The chemical constituents of the thyroid gland. II. The specific physiological activity of certain constituents of the thyroid gland. Proc. Soc. Exp. Biol. Med., 10, p. 165. Kendall, E. C., 1915. A Hethod for die Decomposition of the Proteins of the Thyroid, With a Description of Certain Constituents. J. Biol. Chem., 20, pp. 501-509. Kendall, E. C. 1919. Isolation of the Iodine Compound which Occurs in the Thyroid. J. Biol. Chem. 39, pp. 125-147. Leland, J. P., and Poster, G. L. 1932. A Method for the Determination of Thyroxine in the Thyroid. J. Biol. Chem. 95, pp. 165-179. Monroe, R. A. and Turner C. W. 1949. The Metabolism.of Thyroxine. Lb. Agr. Exp. Sta. Res. Bul. 446. Palmer, W. W., and Leland, J. P. 1935. Comparative calorigenic action of normal and pathological thyroid glands administered in equi- thyroxine doses. J. Clin. Invest. 14, pp. 619-631. Pitt-Rivers, R., and Lerman, J. 1948. The Physiological Activity of the Optically Active Isomers of Thyroxine. The Journal of Endo- CFiflOlogy 5, pp. 223-228. Plummer, H. S. 1921. Interrelation of Function of the Thyroid Gland and of its active agent, Thyroxine, in the Tissues of fine Body. J. A. M. A. 77, p. 243. Rapport, D. and Canzanelli, A. 1931. A comparison of the effects of tyrosine, diiodotyrosine, diiodothyrcnine and thyroxine upon metabolism. Am. J. Physiol. 97, p. 553. Reineke, E. P., Williamson, M. B., and Turner, C. W. 1943. The Effect of Progressive Iodination Followed by Incubation.at High Tempera- ture on fine Thyroidal Activity of Iodinated Proteins. J. Biol. Chem. 147, pp. 115-119. Reineke, B. P., and Turner, C.'W. 1945. The relative thyroidal potency of l- and d,1-thyroxine. Endocrinology 36, pp. 200-206. 4.0- Reineke, E. P., Turner, C. J., Yohler, G. 0., Soover, T. 9., and Beezley, N. P., 1945. Quantitative determination of thyroxine in iodinated casein having thyroidal activity. J. Biol. Chem. 161, pp. 599-611. neineke, E. P., hallach, D. P., and Walterink, L. F. 1949. Thyrox content of synthetic thyroprotein as determined by radioactiv isotope dilution technique. 45 Annual fleeting, Am. Dairy Sci. Assoc. 1950. Cornell Univ., Ithaca, F. Y. Salter, W. T., Lerman, J., Veans, J. H. 1935. The Calorigenic Action of d- and 1-Thyroxine. J. Clin. Invest.14, pp. 37-39. Schittenhelm, A., and Eisler, B. 1932. Uber die Resorption des Thyroxins nach oraler zufuler. Ztschr. exp. Wed. 80, p. 569. (From Monroe, R. A. and Turner, C.'T. 1949. The metabolism of thyroxine. We. Aer. BXp. Sta. Res. Bul. 446). Taurog, A. and Chaikoff, 1946a. The Determination of Thyroxine in the Thyroid Gland of the Rat. J. Biol. Chem. 163, pp. 323-328. Thompson, W. 0., Thompson, P. K., Dickie, L. F. N. and Alper, J. TS 1933. Effect of alkali on the absorption of thyroxine from the gastro-intestinal tract. Arch. Int. Vec. 59 pp. 809-820. U94, Williams, R. J. and Kirby, H. 1948. Paper chromatography using capillary ascent. Science, 107, pp. 481-483. - -41- APPENDIX Purification of Diiodothyronige The diiodothyronine obtained from.Hoffmann-La Roche and Company, was dissolved in hot ethyl alcohol with the aid of 1.5: HCl and pre- cipitated by the addition of one-half saturated sodium acetate solution. The hot mixture was centrifuged and the white crystalline product washed with acetone. A second partial precipitation of the diiodothyronine was carried out by the same method. The purified preparation was stored under refrigeration in a desiccator jar until ready for use. -42— 4 Kilo "Toppert" Stock Ration Yellow corn meal (Thomn)..................o.........oo.....1400 Ground “11019 "fl-1&1; (Thomn)...........0.0.0....IOCOOOOOOOOOO1000 .3 Whole milk powder (corden).................................. Linseed oil meal (Thoman)................................... Alfalfa leaf meal (Thoman).................................. Brewer's Yeast (Strain G) (A. Busch)........................ Taljle salt (iOdized)OOOOOOOO000......OOOOOOOOOOQOOOOOOOOOOOO 800 1:00 40 CHEMISTRY LIBRARY * T612.015 268375 F899 Friedberg. A critical analysis of t . butyl alcohol attraction technique used in the quantiu .tetive determinationof - 1293 02446 7296