THYRSACTEVE ECELENATEQ‘: F‘EQYEENS: I [MMUNQLGGK H TR'fRQID HQRMQNE ANALY$E§ Thesis Fm- ?kc Dogma 0»? Bk. 3. MECHEGRN STATE UNIVERSITY Terrence Wynn Mis‘chier 1967 in 5.3), S This is to certify that the thesis entitled THYROACTIVE IODINATED PROTEINS: I IMMUNOLOGY, II THYROID HORMONE ANALYSIS presented by Terrence Wynn Mischler has been accepted towards fulfillment of the requirements for Ph D degree inlhyainlogy Major professor DateSfipLemheL 1 9, ] 967 0-169 LIBRARYH Michigan State , University I? ABSTRACT THYROACTIVE IODINATED PROTEINS: I IMMUNOLOGY, II THYROID HORMONE ANALYSIS by Terrence Wynn Mischler Thyroactive iodinated casein has been injected and fed to animals for increasing their thyroid activity ever since its synthesis some twenty-five years ago. Yet neither its immunological properties nor the concentration of all its thyroid hormones have ever been determined. These prOperties of a commercially synthesized iodinated casein, Protamone, were investigated using immunological double diffusion and thin—layer chromatography. Protamone contains at least one antigenic component found by diffusing anti-Protamone against it in double diffu— sion analysis. This antigen was called the Protamone antigen. It and two others were observed when anti-Protamone was diffused against casein. Thus Protamone produced antibodies against three antigens of casein origin in Protamone. How- ever, anti-casein diffused against Protamone would result in g9 precipitation and when diffused against casein gg_precipi— tation of the Protamone antigen was seen. Thus, the Prota- mone antigen when present in casein was unable to produce Terrence Wynn Mischler antibodies. Iodinated casein prepared in the laboratory con- tained at least two antigens of casein origin. Proteins extracted from rat thyroid glands contained two thyroid specific antigens. They are believed to represent the 19S and 27S proteins known to exist in the gland. There was, with one exception, no cross reaction between any of the anti-iodinated protein sera and their respective proteins. However, when anti-Protamone was diffused against iodinated casein the Protamone antigen was again observed. Therefore, the Protamone antigen was present in Protamone, casein and iodinated casein, yet it produced antibodies only when in Protamone. Hence it is concluded that the process of Protamone synthesis altered the antigen such that it was able to produce antibodies. This alteration was not involved with the iodination process since the iodinated casein prepared in the laboratory did not contain the altered antigen. Finally, anti-Protamone demonstrated the presence of the Protamone antigen in a deficient commercial thyroid preparation sus- pected of containing poorly iodinated casein. Two-dimensional thin layer chromatography of Protamone hydrolysates demonstrated at least twenty-two components in the acidified n-butanol soluble fraction. There were only five components remaining when the extract was washed with 4N NaOH - 5% Na2C03. Two of these fiye components were identified as thyroxine and triiodothyronine by a number of Terrence Wynn Mischler independent criteria. The concentration of these two hormones was determined to be 0.79% thyroxine and 0.61% tri- iodothyronine. This is biologically equivalent to 3.28% thyroxine. The combined activity of thyroxine and triiodo- thyronine in Protamone and similar iodinated proteins as calculated from this analysis, is sufficient to account for thyroidal activity values obtained earlier from biological assays in mammals. Since this chemical assay is sufficient to account for all biologically determined thyroidal activity, it is unlikely that there are other thyroid active compounds in iodinated casein which have thyroid activity in mammals. This was the first time that a complete analysis of all thyroidal hormones has been accomplished on a thyroid active iodinated protein synthesized ig_vitro. THYROACTIVE IODINATED PROTEINS: I IMMUNOLOGY, II THYROID HORMONE ANALYSIS BY Terrence Wynn Mischler A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology '1967 ACKNOWLEDGMENTS I would like to express my gratitude to Dr. E. Paul Reineke, Professor of Physiology, for all his aid and guid- ance throughout my years as a graduate student and especially for his advice to me on this project. I am grateful for the fine photographic work done by Dr. Jack R. Hoffert, Assistant Professor of Physiology. I appreciate the help and suggestions of Mrs. Judianne Anderson, Physiological Technician, on the chemical analyses used in this research. I would like to thank Dr. Lester Meister, United States Veterans Administration Hospital, Long Beach, California, for supplying commercial thyroid preparations, suspected of containing iodinated casein, for assay by the immunological method reported herein. I would like to express my appreciation to Mr. Bruce Varney, President, Agric-Tech Inc., Kansas City, Missouri, without whose assistance this thesis could not have been completed. Finally, I would like to thank my wife JoAnn for her help and advice to me during my graduate school career. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . 3 Iodinated Casein. . . . . . . . . . . . . . . . 5 Properties of Rat Thyroid Protein . . . . . . 9 Thin-Layer Chromatography of Iodinated compounds 0 O O O O O O C O O O O O O O O O 11 Relative Potencies of Thyronine to its Analogues. . . . . . . . . . . . . . . . . 11 MATERIALS AND METHODS. . . . . . . . . . . . . . . . 14 Starting Materials. . . . . . . . . . . . . . . 14 Preparation of Antisera . . . . . . . . . . . . 17 Immunological Double Diffusion. . . . . . . . . 20 Thin-Layer Chromatography (TLC) . . . . . . . . 22 Hydrolysis of Iodinated Proteins. . . . . . . . 50 RESULTS 0 O O O O O O O O O O O O O O O O O O O O O O 35 Double Diffusion Analysis of Iodinated Proteins 55 Purity of Commercial Iodinated Thyronines . . . 4O Iodinated Compounds in Protamone Hydrolysates . 42 DISCUSSION . . . . . . . . . . . . . . . . . . . . . 57 SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . 67 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 71 APPENDICES . . . . . . . . . . . . . . . . . . . . . 75 iii LIST OF TABLES TABLE Page 1. Summary of Serological Components in Analyzed Proteins. . . . . . . . . . . . . . . . . . . . 57 2. Average Rf Values of Iodinated Compounds Using TLC on Cellulose. . . . . . . . . . . . . . . . 41 5. Percent Concentration of Thyroxine and Triiodo- thyronine in Protamone. . . . . . . . . . . . . 56 iv LIST OF FIGURES FIGURE 1. 10. 11. 12. 2D Thin Layer Chromatograph of Iodinated Compounds. . . . . . . . . . . . . . . . . . Double Diffusion Analysis of Iodinated Proteins O I O O O O I I O O I O O O O O O 0 1D Thin Layer Chromatograph of Protamone Hydrolysate. . . . . . . . . . . . . . . . . 2D Thin Layer Chromatograph of Unwashed Protamone Hydrolysate. . . . . . . . . . . . 2D Thin Layer Chromatograph of Nagcoa-NaOH Washed Protamone Hydrolysate . . . . . . . . 2D Thin Layer Chromatograph of the Acid Insoluble Fraction of Protamone Hydrolysate. 2D Thin Layer Chromatograph of Thyroid Protein Hydrolysate. . . . . . . . . . . . . . . . . Page 27 58-59 44 46 48 51 53 LIST OF APPENDICES APPENDIX Immunological Double Diffusion Buffer. . Protein Stain . . . . . . . . . . . . . . FFCA Iodine Specific Spray. . . . . . . . Pauly's Reagent . . . . . . .~. . . . . . Standard Curves for Hormally Bound Iodine vi Page 76 76 76 76 77 INTRODUCTION The in_vitro synthesis of a thyroid active iodinated protein was accomplished by Dr. E. Paul Reineke some twenty— five years ago. The use of a properly buffered casein solu— tion and incubation at relatively high temperatures, after addition of the correct amount of iodine, resulted in the thyroid active protein called iodinated casein. It was shown, after vigorous hydrolysis, that L-thyroxine could be isolated from this substance. However further research has led a few investigators to suspect that other iodinated thyronines, including triiodothyronine, may also be synthesized during the iodination of casein. Assays of iodinated casein for its thyroxine content have shown considerable variation in their values. Therefore, the true total biological activity in terms of thyroxine content is, at this time, not really known. There is no information regarding the immunological properties of synthetic thyroid active iodinated caseins and their immunological relationships, if any, to naturally occurring thyroid proteins. These synthetic proteins are fed and/or injected into animals as a protein—-yet no data exists on their antigenicity. It was decided to concentrate this investigation on the commercial iodinated casein--Protamone since it is a standardized product readily available and is widely used to increase thyroid activity of animals. The purpose of the research reported herein was two- fold. First, to study the immunological properties of Protamone and compare them to those of two other iodinated proteins; namely, rat thyroid protein and a laboratory- prepared iodinated casein. The second purpose was to obtain an analysis of all thyroid active compounds that exist in Protamone. Therefore, the hydrolysates of Protamone were fractionated and the compounds known to have thyroid activity identified. At this time an attempt will be made to deter- mine the concentration of these compounds and then if this is successful the total thyroid activity of Protamone in terms of hormone concentration will be established. REVIEW OF THE LITERATURE Iodinated Casein With the isolation of thyroxine by Kendall (1915) and its synthesis by Harington and Barger (1927), there were numerous attempts to form thyroxine in_yi££9_by iodination of proteins. The publications concerning these early at- tempts have been thoroughly reviewed by Reineke (1942a, 1946, 1949). The attempts at in_yit£9_synthesis were, in general, not successful but considerable information was obtained on methods and conditions of iodination. Those claims of success were met with skepticism since assays of early products were questionable. Crystalline thyroxine was first isolated from a protein iodinated ig_yi££9_by Ludwig and von Mutzenbecher (1959) and verified by Harington and Pitt-Rivers (1959). There was considerable research in the 1940's by Reineke and co-workers on the conditions needed for iodination of proteins. Reineke and Turner (1942a) found that a bicarbonate buffer, above pH 7.0, was needed to produce a thyroid active iodinated casein. Reineke, Williamson and Turner (1942b) in— vestigated the progressive iodination of casein and found that maximum activity was obtained when 2 moles of iodine per mole of tyrosine was used for iodination. Up to this time all research on protein iodination had been conducted at 580C, considered optimal for thyroxine formation. But in 1945a Reineke, Williamson and Turner iodinated casein with progres- sive amounts of iodine at higher temperatures (700C for 20 hours) and, in addition to confirming their earlier report regarding concentration of iodine per tyrosine, they obtained a fourfold increase in activity when incubating at 700C in- stead of 58°C. This highly active iodinated casein in the tadpole assay was found to have an apparent 8-11% thyroxine content when compared to a D,L-thyroxine standard. Since Reineke and Turner (1945c) determined that D-thyroxine had no activity this value of 8-11% thyroxine should be halved and thus equals 4-5§% thyroxine. Crystalline thyroxine was isolated from iodinated casein by Reineke and Turner (1945b) after hydrolysis with 40% barium hydroxide. It was identified by its characteristic crystalline structure, ultraviolet absorbtion curve and iodine content. The yield was 0.424%, whereas, if the acid insolu- ble fraction of iodinated casein was considered to be all thyroxine, then the yield increased to 2%. Thyroxine is in- soluble at pH 4.5 as first determined by Kendall (1915). Reineke and Turner (1945c) isolated L-thyroxine from iodinated casein by acid hydrolysis and found it had twice the potency of D,L-thyroxine obtained by barium hydroxide hydrolysis. This indicated that barium hydroxide hydrolysis produced racemation of thyroxine resulting in a D,L—mixture. Reineke, Turner, Kohler, Hoover and Beezley (1945a) used both chemical and biological assays to determine the thyroxine content of iodinated casein. The foundation of the chemical assay was laid by Leland and Foster (1952) in which they extracted hydrolyzed thyroid glands with n-butanol, then 1N NaOH and considered the iodine in this extract to be thy— roxine. Blau (1955) modified this method by first extracting the hydrolyzed gland with acidified n-butanol and then wash- ing this solution with a mixture of 4N NaOH and 5% Na2C03. The iodine content of the washed extract was considered to be thyroxine. Reineke §£_al. (1945a) applied the Blau method. to iodinated casein, using 40% barium hydroxide for hydrolysis and found it contained n-butanol soluble iodine equivalent to 5.04% thyroxine. They also demonstrated that diiodotyro- sine had no appreciable thyroxine activity and that a loss of only 7% thyroxine occurred during hydrolysis. There are many other chemical assay procedures and the reviews of Pitt- Rivers (1950) and Barker (1962) should be consulted for more information. Bioassays, on iodinated casein, were also con- ducted by these investigators using the guinea pig C02 production method described by Reineke and Turner (1942a). A 2.79% thyroxine content was found, which closely agreed with the chemical assay of 5.04%. Finally, Reineke and Turner (1945b) found that the addi- tion of any one of a number of manganese oxides, to the incu— bation mixture, would further increase the potency of the iodinated casein to 5.57%, as determined by their chemical assay. Friedberg (1951), using paper chromatography investi- gated the Blau extraction method when applied to hydrolysates of iodinated casein. He demonstrated that two other compounds, besides thyroxine, were present in the washed n-butanol ex- tract of hydrolyzed iodinated casein. It was then apparent that an error was introduced into the thyroxine assay of iodinated casein using the Blau method, because two compounds besides thyroxine were included in the assay. Reineke (1954) employed a highly Specific isotope dilu— tion technique and found a 1.04% thyroxine content in iodinated casein. This compared to a 5.24% thyroxine content obtained by the Blau chemical analysis on the same material. The 1.04% thyroxine content was considered to be a very accurate estimate of the thyroxine content of iodinated casein, but it is too laborious to be used as a routine ana- lytical procedure. One assay has been published on the commercial iodinated casein——Protamone, by Turner and Bauman (1962). They em- ployed the thyroxine substitution method in rats to biologic- ally assay this substance. These workers modified the usual procedure by daily injecting 400 ug of tapazole per 100 gm. of rat. They determined a mean thyroxine secretion rate value by subcutaneous thyroxine injections and then using the same rats determined the value obtained by injecting Protamone instead of thyroxine. They obtained a mean of 1.40% thyroxine equivalent in Protamone, whereas the manufacturer's value was 1.00% thyroxine. However, this publication did not indi— cate the dosage or number of protamone injections given to the rats. Nor was any evidence presented demonstrating that a steady state equilibrium was obtained during the period of Protamone injections. There have been few attempts to demonstrate that com- pounds other than thyroxine are present in iodinated casein. Hird and Trikojus (1948) employed paper chromatography to find two compounds, other than thyroxine, in iodinated casein._ One of these appeared to be diiodothyronine and the other was believed to be triiodothyronine because of its position be- tween thyroxine and diiodothyronine. Friedberg (1951) also using paper chromotography found at least 10 iodinated components in the n-butanol soluble fraction of hydrolyzed iodinated casein. As discussed above, three compounds remained in the alkali-washed extract of hydrolyzed iodinated casein, one of which appeared to be thyroxine. This is the only published evidence that Blau's extraction is not Specific for thyroxine values in Protamone. There has been limited research on the structural and/or immunological properties of non-thyroidal proteins iodinated in_yi§g9, Wormall (1950) is the only investigator of immunological properties of non-thyroidal iodinated proteins that was found in the literature. He demonstrated by a test tube precipitation test that iodinated serum pro- teins of a number of mammals lost their species specificity and that a new specificity characteristic for iodoproteins was produced. He believed that 5:5 diiodotyrosine was responsible for this change. Kamal and Turner (1951) investigated the electrophoretic properties of iodinated casein using the Tiselius apparatus. They demonstrated that non-iodinated casein consisted of an alpha and beta component, whereas iodinated casein had a single component with a mobility slightly faster than either alpha or beta casein. Casein incubated at 700C, without added iodine, also showed the same electrophoretic pattern. Thus it appeared that incubation and not iodination produced this change in electrophoretic patterns. Finally, Williams, Meister, Faircloth and Florsheim (1964) have suggested the possibility that a poorly iodinated casein was added to defective foreign commercial desiccated thyroid preparations that were deficient in thyroidal activity. This could increase the organically bound iodine content enough to meet the only U.S.P. requirement; namely, 0.2% organically bound iodine, and consequently decrease the thyroid activity to even less than that of the original preparation. These conclusions were based on P/N and iodotyrosine/iodo- thyronine ratios which were higher than in a standard active thyroid preparation. These high ratios were found both in a mixture of laboratory—prepared poorly iodinated casein with a known active thyroid preparation and the defective thyroid preparations. These workers also found by ion-exchange chromatography that 20% of the total iodine in the commer- cial iodinated casein, Protamone, was in the triiodothyronine and thyroxine fraction. Properties of Rat Thyroid Proteins There has been considerable research on the biochemical and immunological properties of thyroid proteins. The bio- chemical characteristics have been reviewed by Robbins and Rall (1960), Edelhoch (1965), Edelhoch and Rall (1964) and the immunological properties have been reviewed by Belyavin (1964). However, almost all the published biochemical in- vestigations have used bovine, porcine or human thyroid pro- teins and the immunological investigations have been directed almost exclusively toward research on the problem of auto- immune diseases in the human. The proteins of the rat's thyroid have received little attention. Lachiver, Fontaine and Martin (1965) labeled thy- roid proteins ig_yi§£9 with 1131, then waited varying lengths of time before removing the glands and extracting the pro— teins. They separated the proteins by sucrose gradient centrifugation and found three protein fractions with sedi- mentation coefficient of 12S, 19S and 27S; the 19S fraction correSponded to thyroglobulin. The I131 associated with the 12S fraction reached 4-5% of total protein-bound 1131 after 4—5 hours and then decreased with time. On the other hand, 10 1131 associated with the 27S fraction increased progressively with time and contained about 10% of total protein bound 1131 1131 was bound to the after 52 hours. The remainder of the 19S fraction. Robbins, Salvatore, Vecchio and Vi (1966) have investi— gated the time course of iodination of rat thyroid protein by "equilibrium" and "pulse" labeling. Thyroglobulin (193) was labeled at a faster rate than the 27S iodoprotein, but the 278 protein contained a higher concentration of labeled con- stituents. Thus the 278 protein was found to be a major storage site of thyroid hormone even though it may only consti— tute 10% of the total protein. Also shown by these authors was an ultracentrifugal heterogeneity of thyroglobulin, in which a slower-sedimenting fraction, containing less iodine, was separated from a more highly iodinated faster—sedimenting fraction. It was believed that the slower component was "newly synthesized" thyroglobulin whereas the faster component might be "old" thyroglobulin. The only immunological investi- gations of thyroid saline extracts where auto-immunity was not being studied, was carried out on beef and hog thyroids by Perelmutter and Stephenson (1964). They observed that 19S (thyroglobulin) and 278 components gave rise to two precipi— tation bands when diffused against anti-thyroid serum by the immunodiffusion techniques. Immunoelectrophoresis indicated that these antigens had mobilities characteristic of alphag globulins. They concluded that the 19S and 278 components appear to have different immunochemical properties. 11 Thin-Layer Chromatography (TLC) of Iodinated Compounds West, Wayne and Chavre (1965) were the first to separate iodinated tyrosine and thyronines using TLC. They had limited success employing silica gel as a supporting medium for separation and identification of L-thyroxine and its metabolites in both plasma and bile. Milstien and Thomas (1965) separated the thyroid hormones and iodide using cellulose as their medium. They described a method for analyzing the iodide content of the cellulose area containing the separated compounds. The method of Faircloth, Williams and Florsheim (1965) was able to separate all naturally occurring thyroid com— pounds, iodide and 5:5 diiodothyronine, using two dimensional development on a cellulose medium. One solvent was formic acid/H20 1:5 and the other was T-butanol/2N NH30H/chloroform 576:70:60. They also reported a procedure for iodine analysis of the chromatographed compounds. Relative Potencies of Thyroxine to Its Analogues The literature abounds with publications and reviews regarding the ig_yi£gg and ig.yiyg synthesis, isolation, metabolism, biochemistry and biological properties of thyroxine and its analogues. The text edited by Pitt-Rivers and Trotter (1964) and the reviews by Roche and Michel (1956), Mayo Clinic Proceedings Vol. 59, No. 8 and DeGroot (1965) should be con— sulted for this information. Assays of thyroxine have already 12 been discussed above. The concern of the review presented in this thesis is the relative potencies of thyroxine to its analogues, since the bioassay of iodinated casein was based on metabolic responses resulting from injections of iodinated casein, with thyroxine used as a reference com- pound. Roche and Michel (1956) have reported that four iodi- nated thyronines were found in the thyroid gland: (1) L- thyroxine, (2) L-5:5:5' triiodothyronine, (5) L-5,5',5' tri— iodothyronine and (4) L-5,5' diiodothyronine. These authors state, that number 4 was only slightly less active than thy— roxine. However, Stasilli, Kroc and Meltzer (1959) were un- able to confirm this report and believe that the L-5:5' diio- dothyronine of Roche g£_al. (1956) was contaminated with L-5:5:5' triiodothyronine. Stasilli gt_al, (1959) also re- ported no appreciable calorigenic activity in 58 of 40 thy— roxine analogues tested in rats. The only two having any appreciable activity were the naturally occurring thyroid hormones, thyroxine and L-5:5:5' triiodothyronine. It there- fore appears that triiodothyronine and thyroxine are the only iodinated compounds which have any significant biologi— cal activity, at least when tested in the rat. Finally no publication besides that of Roche §£_§l, (1956) was found which describe any iodinated thyronines in the thyroid gland other than thyroxine and triiodothyronine (see Pitt-Rivers and Rall, 1961). 15 There is considerable difficulty in the determination of relative potencies of thyroxine versus triiodothyronine because of the differences in plasma protein binding and bio- logical half-lives of these two hormones. Gross and Pitt- Rivers (1955) found that triiodothyronine goiter prevention activity was five times greater than thyroxine using a molar basis of comparison. Heming and Holtkamp (1955) used thy— roidectomized rats to compare the calorigenic activities of triiodothyronine and thyroxine. They found that triiodothy— ronine was 5.5 times more potent than thyroxine using a molar basis of comparison. Heming and Holtkamp also found this same ratio when the goiter prevention potencies of these two hormones were determined. Stasilli et al. (1959) injected triiodothyronine and thyroxine for 14 days in rats and at the same time determined their metabolic rates from day zero until return to control level. These workers found that tri— iodothyronine had 8 times the calorigenic and goiter preven- tion potencies of thyroxine using a weight basis for compari- son. Finally Reineke and Lorscheider (1967) found that triiodothyronine had 4.09 times more activity per unit weight than thyroxine, as shown by the thyroxine substitution method of Reineke and Singh (1955). MATERIALS AND METHODS StartinQVMaterials A. Protamone The commercially synthesized iodinated casein Protamone (Lot No. 1114)* was supplied by Agri-Tech, Inc. of Kansas City, Missouri and used throughout these investigations, except for one occasion, where Lot No. 1676 was obtained for comparison with Lot No. 1114. A 2.5% solution of each sample was prepared by dissolving the required amount in NaHC03 solu? tion at pH 8.0 (700 mg NaHCOa in 100 ml of distilled H20) and then stored at -200C until used. The commercial procedure of preparing Protamone is unpublished; however, the principal difference between the laboratory method used for synthesis of iodinated casein and the method for Protamone synthesis was the handling of the acid precipitated iodinated protein. Protamone is placed in a vacuum rotary dryer at 15-20 pounds per square inch steam pressure at 25 inches of vacuum until dried. The laboratory prepared iodinated casein was lyOphil— ized and stored at -2OOC. * Thyroxine content of Lot No. 1114 was stated to be 1.07% in the certificate of analysis issued by the manufacturer Hoffman—Taff, Inc., Springfield, Missouri. 14 15 B. Bovine Casein Bovine casein was obtained by acidifying skimmed non- pasteurized bovine milk to pH 4.6 by the addition of 1N HCl. The precipitated casein was washed four times with acidified distilled water, lyophilized and stored at -200C. Commercial casein, used to synthesize Protamone was obtained for com- parison with the laboratory-recovered casein. A 5.0% solu- tion of each protein was prepared as previously described for solutions of Protamone. C. Iodinated Casein Iodinated casein was synthesized in the laboratory according to the method of Reineke §£_al. (1945a). Five gms of NaHCOa was added to 700 ml of distilled water, then 20 gms of laboratory prepared casein was mixed into this buffered solution. This casein solution was heated to 40-450C in a water bath and 5.7 gms of powdered iodine was added slowly with constant stirring. This iodine-casein solution was mixed for 1.5 hours, then incubated at 65-70°C for 20 hours with vigorous stirring. The iodinated casein was precipitated at pH 4.5 with 1N HCl, washed 4 times with acidified water, lyophilized and stored at -200C. A 5% solution was prepared as previously described for Protamone. Chemical assay of this compound by Hoffman-Taff, Inc., indicated a 0.45% thyroxine content. Also this compound, when placed in the food of 7 Holtzman rats at a level of 0.075%, resulted in a twofold in- crease in the 02 consumption of these rats over 7 control rats. 16 This difference was significant at the P level of 0.01 using a one-sided paired T-test. These two assays indicated that the compound had sufficient thyroidal activity for use in subsequent investigations. D. Rat Thyroid Protein The thyroids from 120 rats were removed, trimmed and frozen at -200C. These thyroids were homogenized in cold 0.85% NaCl at one gland per 0.5 ml of saline. The extracted proteins were centrifuged and the total volume of supernatant was recovered. This supernatant was lyophilized and stored at -20°C. The protein concentration of supernatant was calcu? lated to be 1.16% and was maintained whenever the protein was redissolved in distilled water. E. Commercial Thyroid Preparation Possibly Containing Iodinated Casein The Review of Literature indicated that Dr. L. Meister has evidence which suggests that some commercial thyroid preparations may have been adulterated with an iodinated casein compound. Seven coded preparations supplied by Dr. L. Meister were tested by an immunological procedure to de— termine if evidence of adulteration could be found. They were dissolved in a NaHCOa solution at pH 8.0 at a starting concentration of 2%. A considerable amount of each sample failed to go into solution; therefore centrifugation was used to clarify the solutions. The supernatant was frozen at -20°C until used. 17 Preparation of Antisera Two different methods were used for the formation of antiserum against the different compounds. One was the use of the alum precipitin technique, the other the use of Freund's complete adjuvant. The preparation of the antigen- adjuvant mixtures was, with one exception, the same for formation of all antisera. Therefore the general procedure for preparing these mixtures will be described below. a A. Freund's Complete Adjuvant Method Equal volumes of Freund's complete adjuvant (Difco Laboratories) was emulsified with the antigen solution in a Waring Blender. The water-in-oil emulsion was considered to be stable when a drop of it placed on cold tap water remained intact. The emulsions were stored at 10°C while being used. B. Alum Precipitin Method The proportions of constituents used to prepare this antigen-adjuvant mixture are described below and can be ad— justed to give the desired total volume. One ml of the anti- gen solution was mixed with 5.2 ml of distilled water. Then 5.6 ml of 10% alum (Potassium aluminum sulphate — KA1(SO4)2' 12H20) was added and pH adjusted to 6.5 with 5N NaOH. The sediment which resulted was washed 5 times with isotonic saline (containing Merthiolate 1:10,000) and made up to a volume of 4 ml with this solution. The one exception was the preparation of alum-precipitated rat thyroid protein, where 18 because of the low concentration of protein (1.16%), equal volumes of all constituents were employed. All of the antigen-adjuvant mixtures were stored at 10°C prior to being injected. Dutch black-belted rabbits were used for antisera production. Control bleedings were taken prior to immuni- zation, and at completion of the injection schedule the animals were bled again--both times by cardiac puncture. The blood was allowed to clot at room temperature for one hour and then stored overnight at 10°C. The serum was then decanted, centrifuged and frozen at -200C until used. The following are the compounds to which antisera were prepared, the adjuvants employed and the immunization schedule used for each antiserum. A. Rabbit anti-Protamone (abbreviated anti-Protamone) 1. Freund's complete adjuvant method One ml of the emulsified antigen was injected subcu- taneously in five abdominal sites on the first and third week. The second week one ml was injected intramuscularly in the thigh. The fourth week one ml was injected intraperitoneally and the fifth week the animals were bled. 2. Alum precipitin method Two ml of the alum-precipitated antigen was injected intramuscularly once a week for five weeks in the thigh of each rabbit. On the third week one ml was injected into each of two foot pads. The rabbits were bled on the sixth week. 19 B. Rabbit anti-Lyophilized bovine casein. (The casein prepared in the laboratory as described earlier was used as anti- gen.) 1. Freund's complete adjuvant method One ml of the emulsified antigen was injected subcu- taneously in five abdominal sites in each rabbit on the first and second week. The third week one ml was injected intraperitoneally and the rabbits were bled on the fifth week. 2. Alum precipitin method Two ml of the alum-precipitated antigen was injected intramuscularly in the thighs of each rabbit once a week for seven weeks. Also one ml was injected into each of two foot pads on the third week. Rabbits were bled on the eighth week. C. Rabbit anti-iodinated casein. (The antigen was Lyophilized iodinated bovine casein prepared as described earlier.) Anti-iodinated casein was prepared by the alum precipitin and Freund's complete adjuvant method in the same way as described for formation of anti-casein. D. Rabbit anti-rat thyroid protein (abbrevi- ated anti-thyroid protein) 1. Alum precipitin method Two ml of the alum precipitated antigen was injected intramuscularly into the thighs of rabbits once a week for three weeks. The rabbits were bled on the fourth week. 20 Antibodies directed against any rat serum proteins contami- nating the thyroid protein preparation were removed by the mixing of anti-thyroid protein with rat serum. This resulted in absorbed anti-thyroid protein. E. Rabbit anti-bovine blood serum (abbreviated anti-bovine serum) 1. Alum precipitin method Two ml of the alum precipitated antigen was injected intramuscularly into the thighs of rabbits once a week for four weeks. The rabbits were bled on the fifth week. F. Rabbit anti-rat blood serum (abbreviated anti-rat serum) 1. Alum precipitin method The immunization schedule was the same as that used to produce anti-thyroid protein. Immunologicalngubleggiffusion The basic theory of double diffusion was developed by Ouchterlony (1958). A layer of buffered agar, usually in a petri dish, has most often a series of 4 wells (holes) arranged in a circle around a center well. The antiserum is deposited in the center well and the antigens in the outer wells. This can of course be reversed as the need arises. There is a diffusion of antigen and antibody toward each other in the agar with a meeting of the two diffusion fronts which result in a zone of optimum proportions somewhere in the overlapping area. Antigen-antibody precipitation occurs 21 and a white precipitin line appears in the agar, thus indi- cating that at least one antigen-antibody system is present. Crowle (1961) gives a complete review of all types of immuno- diffusion methods and should be consulted for further details. The following procedure was developed for double dif- fusion. Five ml of 1% Oxoid Ion agar No. 2 (Colab No. L12) prepared in phosphate buffer at pH 7.4, ionicity 0.15 (con- taining Merthiolate 1:10,000), was pipetted onto a 2 x 5 inch glass slide (see Appendix 1 for buffer formula). After the agar gelled the slide was laid on a piece of paper, which had drawn on it the appropriate well pattern, and the wells were cut with a 10 mm cork borer. The agar was removed using curved forceps, the wells charged with suitable reactants, and left to diffuse and precipitate in a humid atmoSphere for four days at 250C. The agar was washed in frequent changes of distilled water for 5 days to remove the non-precipitated proteins. Then a moistened piece of Whatman No. 40 filter paper was laid on the agar and the agar was dried to a film over night at room temperature. Staining was done with Amido Schwarz (see Appendix 2 for formula) and destaining accom- plished with 2% acetic acid. Two different well patterns were employed. The first consisting of four wells arranged in a circle 5 mm from the center well, was used for most of the immunological experi- ments. Second, when the purpose was to compare two antigenic solutions to one antiserum or one antigenic solution to two 22 antiserums, a three well pattern was used. This consisted of 5 wells in a triangular arrangement each 5 mm apart (see Results for illustrations of these patterns). Whenever there was fusion of precipitin lines it was concluded that the anti- gens involved were serologically identical (figures 2, 5, 4, 5, 6 and 7). Thin-Layer Chromatography (TLC) TLC employs a thin layer of supporting medium, usually Spread on a glass plate, for separation of compounds. The compounds to be separated, are deposited on the layer and the solvent is allowed to migrate up the medium resulting in separation. This technique is, in many ways, a modification of paper chromatography. However TLC has the advantage that many different supporting media can be used in conjunction with a variety of solvent mixtures. Absorption, partition and ion-exchange chromatography can be employed separately or in combination, resulting in a more versatile technique than paper chromatography. TLC also has the advantage of faster development time, greater sensitivity and simplicity. The primary disadvantage of TLC is that large amounts of compounds are at times difficult to separate because of the low capacity of the thin layerof supporting medium. The books of Stahl (1965) and Randerath (1966) Should be con- sulted for more information regarding thin layer chroma- tography. 25 The separation of iodinated compounds was accomplished by a thin layer composed of cellulose powder. Fifteen gms of MN 500 HP cellulose (Macherey, Nagel and Co.) was homogen- ized with 90 ml of distilled water in a Waring Blender. This Slurry was spread onto glass plates, using a Spreader and Spreading board manufactured by Research Specialties Co., forming a wet layer 0.250 mm thick. The plates were allowed to dry until they could be handled and then heated at 1100C for 15 minutes, resulting in a dry mat of cellulose powder on a glass plate. Two solvent systems were employed for separation of iodinated compounds isolated from thyroactive proteins (modification from Faircloth, §t_al, (1965)). 1. Formic Acid/water 5:5 2. n-Butanol/ZN NH4OH/chloroform 57:7:6 The developing chambers were lined with filter paper that was then saturated with the solvent, leaving about 1.5 cm of solvent on the bottom of the tank and finally sealed with a cover. It is important that the tanks are saturated at all times so that the solvent front migrates uniformly up the cellulose layer. The iodinated compounds, listed below and followed by their abbreviations were used for reference standards. They were dissolved in acidified n-butanol and stored at 100C. 1. thyroxine 1H(T4) 2. 5:5:5' triiodothyronine (T3) 24 5. 5:5 diiodothyronine (T2) 4. diiodotyrosine (DIT) 5. monoiodotyrosine (MIT) 6. KI (I-) The concentration of these compounds was 0.04 mg/ml, except for KI, where it was 0.4 mg/ml. Mixtures of these compounds were made on a one-to-one basis and used where appropriate. The solutions to be separated were deposited using Drummond micro-pipettes (1 micro-liter for one dimensional and 10 micro-liters for two dimensional TLC). The sample was spotted on the cellulose 2.0 cm from the edge and the solvent evapor- ated to dryness with hot air from a portable hair dryer. If iodine analysis was to be performed on any of the chromo- tographed solutions, the pipette was rinsed 2 times with n-butanol and the rinsings were added to the Spot. This edge was then immersed in the solvent and the tank resealed. When the solvent front reached at least 14 cm, the plate was removed, solvent front marked and the cellulose dried. The location of iodinated compounds was determined by the FFCA iodine-specific spray of Gemlin and Virtanen (1959). The area containing iodine appeared as a deep blue Spot on a light green background (see Appendix 5 for procedure). However these Spots fade rapidly in the light so they must be marked soon after Spraying. This Spray follows the Beer- Lambert law and darkness of Spots is proportional to amount of iodine present. A Spray Specific for benzene rings, 25 Pauly's reagent described by Boock (1952), was used on a limited basis (see Appendix 4 for procedure). The FFCA spray was very sensitive, as spots with a concentration of only 0.001 microgram of hormonal iodine could be detected. However Pauly's Spray could detect concentrations only in the range of 0.10 microgram of hormonal iodine. RF values on one dimensional chromatographs were calculated in the usual manner by dividing the distance the compound migrated from its origin by the distance the solvent front migrated from the origin. One and two dimensional thin layer chromatography was used for separation of iodinated compounds. Figure 1 illus— trates both one and two dimensional thin layer chromatography of all standards listed above. A mixture of all standards was placed at point 1 and appropriate combinations of standards deposited at points 2-7. The chromatogram was de- veloped first in the formic acid/H20 solvent, thus separating the mixture along the edge and also separating the standards 2-4. The plate was dried and then developed at a right angle to the first run with the n-butanol/2N NH4OH/chloroform sol- vent, thus separating this mixture a second time and also separating standards 5-7. The plate was dried again and Sprayed with the FFCA reagent. This results, as seen in Figure 1, in a two dimensional separation of the mixture in both solvents and a one way separation of the standards in each of the solvents. As illustrated for thyroxine a tri— angulation method (Randerath, 1966) was then used for 26 Figure 1. Two dimensional thin layer Chromatograph of iodinated compounds. Column 1: Mixture of all iodinated compounds chromatographed with both solvents. Columns 2-4: Reference standards chromatographed only with the formic acid/water solvent. Columns 5-7: Reference standards chromatographed only with the butanol/NH4OH/chloroform solvent. The triangulation method was used to identify a compound chromatographed two—dimensionally. Two lines were drawn, parallel to the sides of the plate, through the known refer- ence standard chromatographed in each solvent. These two lines will intersect at the location of the same substance on the two dimensional part the chromatograph. The identi- fication of thyroxine using this method is seen in Figure 1. 27 20 THIN LAYER CHROMATOGRAPH OF IODINATED COMPOUNDS @ "g9; {ai'(:) on 5 SOLVENT Q MIT 80‘” O FRONTS .0 Q3 err 069 ® or? O @--___------__ r 6) "22:7 4 3 2 I <——— N-IUTANOL/NH O7/CHLOROFORM Figure 1 28 identification of a compound chromatographed two-dimension- ally. This method involved the drawing of two lines, parallel to the Sides of the plate, through the known refer- ence standard chromatographed in each solvent. These two lines will intersect at the location of the same substance on the two-dimensional part of the chromatogram. If one- dimensional TLC was used for separation of mixtures, standards were chromatographed in parellel columns. Commercially synthesized iodinated thyronines were chromatographed, using the formic acid/water solvent in combination with the highly sensitive FFCA spray, to determine their purity. These compounds, along with their source, are listed below. . L-thyroxine--Glaxo Laboratories, England L-thyroxine--Merck, Sharp and Dohme L-thyroxine--Baxter Laboratories 5,5,5'L—triiodothyronine—-Smith, Klein and French . 5,5,5'L-triiodothyronine-eNutritional Biochemicals . 5,5 L-diiodothyronine-—Glaxo Laboratories, England (DCDUP‘CNNH Their concentrations were 0.04 mg/ml in acidified n-butanol. It will be shown that one dimensional TLC using the formic acid/H20 solvent was adequate for separation of T4 and T3 from Protamone hydrolysates, thus allowing an assay of these hormones to be accomplished. The iodine contents of chromatographed components were determined by a modification of Barker's (1951) procedure for iodine analysis. The loca- tion of the compounds was determined by comparisons to reference standards chromatographed parallel to the mixture of unknowns. Thexcellulose with the unknowns could not be 29 sprayed with the FFCA reagent as it interfered with the analysis. A 1.5 cm square of cellulose, corresponding to 'theilooationcfifthe component, was scraped off the plate and placed into a 40 ml centrifuge tube. At least two blanks of Similar size per individual hormone analysis were removed at the same time. Two ml each of 2N HCl and 7N H2804 were added to all tubes. Tubes containing thyroxine had 5 m1 of water added, whereas tubes with triiodothyronine had just 2 ml of water added because of the lower T3 concentration. The solutions were mixed, centrifuged and 5 ml of the super- natant was placed in matched photometric tubes for iodine analysis. The solutions were equilibrated at 50°C in a water bath whereupon 0.5 ml of arsenious acid (Hycel Arsenious Acid Reagent——0.25% A5203 in 2.5% H2804) plus 0.5 ml of ceric ammonium sulfate (Hycel Ceric Ammonium Sulfate Reagent--0.95% CeCNH4)4(SO4)4) were added. The tubes were incubated at 500C for exactly 20 minutes and then 0.5 ml of 1% brucine sulfate was added. The percent transmittance was read at 480 mu with a Coleman Junior II Spectrophotometer and the results recorded as percent transmittance (%T) minus %T for the cellulose blank. Thyroxine and triiodothyronine standard curves were con— structed by first placing a graduated series of known amounts of these two hormones on cellulose plates, spraying the plates with the formic acid/water solvent and then analyzing the cellulose as described above. The percent transmittance minus the blank versus the total hormonal iodineper spot was plotted 50 on linear graph paper. A different standard curve for thy- roxine and triiodothyronone was obtained and the reason appeared to be that the cellulose was more concentrated in the T4 tubes and affected the analysis. However, if T3 was analyzed the same as the T4 (i.e. addition of 5 ml water in— stead of 2 ml) a curve identical to that for thyroxine was obtained.(Appendix 5). The iodine values of unknowns were read from the appropriate standard curve. The percentage of each hormone in Protamone was determined by dividing the hor- monal iodine in each spot by a conversion factor (0.6555 for T4 and 0.5856 for T3) to obtain amounts of total hormone per spot, then multiplying by a dilution factor of 25,000 to determine the total amount of hormone per hydrolysate. Finally this value was divided by the original weight of the Protamone hydrolyzed and multiplied by one hundred to give the percent of the two hormones in Protamone, calculated on a weight basis. Hydrolysispfjlodinated_§roteins The method of Reineke §£_al, (1945a) was used for hydroly- sis of Protamone. One-tenth gm of Protamone was placed in 15 x 150 mm test tubes along with 0.52 gm of barium hydroxide (Ba(OH)2°8HgO) and 0.64 ml of distilled water. An air reflux condenser was attached and the mixture refluxed for 20 hours in a boiling Water bath. At this time 2.5 ml of distilled water was added and the supernatant decanted into a 60 m1 separatory funnel. The remaining precipitate of barium salts 51 was decomposed by adding 0.2 ml of n-butanol, 0.5 ml of 5.5N HCl and warming. When decomposed the salts were tranSferred into the separatory funnel and this combination made up to a volume of 10 ml with distilled water. The pH of this solu- tion was adjusted to 4.0 with 5.5N HCl. It was then extracted with an equal volume of n-butanol. At this time some hydroly- sates were washed with equal and then half volumes of 4N NaOH containing 5% Na2C03. The final volume of the extracted hydrolysates was made up to 25 ml in either case with n-butanol. The acid n-butanol extract is referred to as unwashed Protamone hydrolysate, whereas it is termed washed Protamone hydrolysate when extracted with the 4N NaOH - 5% Na2C03 solution. The acid insoluble fraction of Protamone hydrolysates was obtained by the method of Reineke and Turner (1945b). The hydrolysis was performed exactly the same as above, except that just 10 ml of water was added to the barium hydroxide solution after completion of hydrolysis. The pH was then adjusted to 4.5 with 5.5N HCl. The resulting precipitate was washed 2 times with acidified distilled water and dis— solved in acidified n-butanol to a volume of 25 ml. Rat thyroid protein was hydrolyzed by the method of Lemieux and Talmage (1966) using 8% barium hydroxide. Ten mg of lyophilized thyroid protein was placed in a 15 x 150 mm test tube and extracted with ether 5 times. One ml of 8% barium hydroxide was added, an air reflux condensor was 52 attached and this mixture was refluxed for 6 hours in a boil- ing water bath. The pH was then adjusted to 1 with 5.5N HCl and the solution was extracted with 1 ml of n-butanol. This extracted hydrolysate was made up to a final volume of 2 ml with n-butanol. RESULTS Double Diffusion Analysis of Iodinated Proteins The results of the immunological investigations of thy- roactive iodinated proteins are summarized in Table 1 and presented in more detail in Figures 2-7. Antiserums produced against bovine and rat serum demonstrated at least 8 antigenic components in their corres- ponding serums. Each anti-serum against Protamone, produced by either of the two immunization methods, demonstrated at least one antigenic component in Protamone. These two components were shown to be serologically identical; thus Protamone contained at least one antigenic component that was referred to as the Protamone antigen (Figure 2). The antiserum prepared by the alum precipitin method also revealed another Protamone com- ponent which appeared as a faint precipitin line (Figure 2) and was found to be of bovine serum origin. The anti— Protamone produced by Freund's adjuvant method gave stronger precipitations and was used for the remainder of the research. Dilutions of 1/10, 1/100 and 1/1000 of the 2.5% Protamone solution resulted in no change in the number of Protamone antigens; only a loss of the one component at 1/1000 Protamone dilution. Protamone Lot No. 1676 reacted the same as Lot No. 1114 when diffused against anti-Protamone, Showing that the 55 54 same serological component was present in both preparations (Figure 5). Each immunization method demonstrated that laboratory prepared casein had at least four antigenic components and they were serologically identical. Again the anti-casein produced using Freund's adjuvant gave the strongest precipi- tin reaction and was used for the remainder of these investi— gations (Figure 4). The same four components, detected in the laboratory prepared casein, were also found in the com- mercial casein (Figure 4). Dilutions of 1/10 or 1/100 casein resulted in no change in the number of serological components, only a loss of them at a dilution of 1/100. Anti—Protamone diffused against either laboratory or commercially prepared casein produced a single precipitin line that was serologically identical to the Protamone com- ponent. Also it was observed that Anti-Protamone contained antibodies directed against two other casein antigens (Figure 5). It must be made clear that the Protamone antigen, demon- strated to exist in casein by anti—Protamone, was not detected when anti-casein was diffused against casein. When anti- casein was diffused against Protamone n2_precipitation occurred, nor did it occur if the 2.5% Protamone, diluted 1/10, 1/100 or 1/1000, was diffused against anti-casein. It appears now that Protamone does not have a completely compound-Specific antigen since anti-Protamone will react with casein. However anti-casein will not react with 55 Protamone. A possible explanation will be presented later in this thesis. Laboratory prepared iodinated casein was observed to contain at least one antigenic component when diffused against anti-iodinated casein serum prepared by the Alum precipitation method and it was found to be of casein origin. The Freund's adjuvant method of immunization did not produce any antibodies to iodinated casein in rabbits. When anti- casein was diffused against iodinated casein two-antigenic components were found to exist in iodinated casein. A bovine serum antigen was found in iodinated casein, but it was not identical to any of the components found using anti-iodinated casein or anti-casein. Thus it appeared that laboratory pren pared iodinated casein could produce no antibodies which were compound specific, and that all antigenic components found in iodinated casein were either of casein or bovine serum origin. Rat thyroid protein was found to contain three antigenic components when diffused against anti-thyroid protein. A rat serum component was found in rat thyroid protein since anti- rat serum could cause one precipitin line. Absorbed anti- thyroid protein was diffused against rat serum and rat thyroid protein at the same time. Two antigenic components Specific to thyroid proteins were observed and also one specific to rat serum was present (Figure 6). It can be seen in Figure 6, that even though the absorption of all antibodies to rat 56 serum protein was not complete, there was sufficient separa- tion of precipitin lines to justify this conclusion. The immunological interrelations, if any, between the thyroactive iodinated proteins were studied using the double diffusion technique. There was no cross reaction of the anti—iodinated casein with Protamone nor with rat thyroid protein. The same was shown when anti—thyroid protein was diffused against iodinated casein and Protamone. There were no precipitation lines formed when anti-Protamone was dif- fused against rat thyroid protein. However when anti- Protamone was diffused against iodinated casein one precipi- tation line was formed and it was serologically identical with the Protamone antigen (Figure 7). Therefore this one Protamone antigen was found to exist in Protamone (Figure 2), in casein (Figure 5) and in iodinated casein (Figure 7). However, only anti—Protamone could demonstrate its presence in any of these three substances. Immunological analyses of unknown commercial thyroid preparation submitted by Dr. L. Meister revealed that two of them contained the Protamone antigen and that anti-casein produced no reaction in any of the preparations. When the identities of the compounds were obtained from Dr. Meister it was learned that one of the preparations which reacted, when diffused againSt antiéPrOtamone,:contained_poorly iodi- nated casein prepared in his laboratory that was mixed with U.S.P. thyroid powder. The other preparation which contained 57 z_w._.omm Qo¢>zk u. ._.z< owmxommd z_m._.omm o_om>x._.u_kz< Zamwm Pom-_._.z< Embomm 90m>IF I 3 mm m z.mmom ZDKNM wz_>om 9 $3254 A. mzmo_kz< mz_m._.omm QMN>4m<223m a magma 58 ANTI-PROTAMONE ANTI-PROTAMONE mruno‘s ALUM PPT. O/Q PROTAMONE Figure 2 PROTAMONE PROTAMONE NOJH4 NOJ676 (>0 ANTFPROTAMONE Figure 5 CASHN'LAB. CASHN'COM. o>0 ANTI-CASEIN Figure 4 59 CASEIN Q PROTAMONE CASEIN O [6%) O Figure 5 RAT THYROID O '0 Figure 6 IODINATED CASE PR°T‘”°“E IomNATED CASEIN UROT Q IODINATED CASEIN Q Figure 7 40 the Protamone antigen, was a defective foreign commercial preparation suspected of containing iodinated casein. There were three other defective foreign preparations which, along with two normal U.S.P. thyroid powders, gave no immunological reaction. Purity of CommercialIodinated Thyronines The average Rf values of chromatographed reference compounds are presented in Table 2. It can be observed that only the formic aciddwater solvent was able to separate the iodinated thyronines tested. However, the other solvent had the advantage of being able to separate DIT from T2. The method of separation and identification of these standards was discussed and illustrated in an earlier section (Figure 1). Because of the extreme sensitivity of the TLC method the purity of commercial iodinated thyronines was determined employing the formic acid/water solvent. The L—thyroxine synthesized by Glaxo Laboratories contained not only thyroxine but also triiodothyronine as a contaminant. The L-thyroxine supplied by Merck, Sharp and Dohme contained not only thyroxine, but also two other compounds, one which migrated Slower than thyroxine and another with the same Rf as triiodothyronine. The Baxter Laboratory L-thyroxine was completely pure as no other iodinated compound than thyroxine could be found. The two preparations of L—triiodothyronine contained impuri— ties. The Smith, Klein and French L-triiodothyronine was contaminated with a small amount of thyroxine. The Nutritional 41 N N. GO. ¢O. Oh. Ob. I». Vb. o b. O s... O b. on. on. C 52331056 :z\._oz<5m- z N o {:3 22:9. mac-54.30 20 01:. 02.9... mozaomxoo ou... um me< N magma 42 Biochemicals Co. L-triiodothyronine was contaminated with thyroxine and with a component which mitrated slightly faster than triiodothyronine. The one sample of diiodothyronine tested contained no other iodinated compounds. Iodinated Compounds in Protamone Hydrolysates One dimensional TLC of unwashed Protamone hydrolysate revealed at least 8 iodinated compounds labeled 0-7 in order of increasing migration rates (Figure 8). The washed hydroly- sate showed only 7 compounds Since number 1 disappeared (Figure 8). However, there were quantitative differences be-_ tween the concentrations of compounds 6 and 7. These two compounds were present in large amounts in the unwashed hydrolysate, but in the washed hydrolysate they were almost absent. Two dimensional TLC of the unwashed Protamone hydroly- sate increased the number of iodinated compounds separated from 8 to at least 22 (Figure 9). MIT and DIT were identified and compounds which corresponded with those labeled 0, 2, 5, 4 and 5 in Figure 8 were also observed. Compounds 6 and 7 listed in Figure 8 were composed primarily of MIT and DIT as seen in Figure 9. Compounds 2 and 4 were identified as thyroxine and triiodothyronine. The evidence for this identi- fication will be presented at a later time. The TLC of the washed Protamone hydrolysate is seen in Figure 10. Almost all iodinated compounds have been removed 45 Figure 8. One dimensional thin layer chromatography of Protamone hydrolysate. Column 1: reference compounds Column 2: unwashed Protamone hydrolysate Column 5: washed Protamone hydrolysate 44 ID THIN LAYER CHROMATOGRAPH OF PROTAMONE HYDROLYSATE F 32:33:11 @608 8 mo 0 30.24» .\ @20 O 2 no O 0“ IL 45 Figure 9. Two dimensional thin layer Chromatograph of unwashed Protamone hydrolysate. Column 1: unwashed Protamone hydrolysate Columns 2-5: regerence compounds 46 20 THIN LAYER CHROMATOGRAPH OF UNWASNED PROTAMONE HYDROLYSATE 0': @63 ~30} new; W 86 5' 0'05” 2 @694 O Q Q 08 é é i 47 Figure 10. Two dimensional thin layer Chromatograph of Na2C03-NaOH washed Protamone hydrolysate. Column 1: washed Protamone hydrolysate Columns 2-5: reference compounds 48 20 rum LAYER CHROMATOGRAPH OF NoOH NA:COs WASHED PROTAMONE HYDROLYSATE DIT m1; . 005 @® -4 (SOLVENT ‘FRONTS MIT 00M” Or® ’5 O ‘0 DIT DIT 3 ® 04. O (3 .. 00 '22:? up ‘0 3. . Figure 10 49 by the washing process except for the five labeled 0, 2(T4), 5, 4(T3), and 5. A trace of MIT, DIT and four others can be seen. It is apparent from Figures 9 and 10 that compounds labeled 0, 2(T4), 5, 4(T3), and 5 are identical in both the washed and unwashed hydrolysates of Protamone. The acid insoluble fraction of the Protamone hydrolysate still contained compounds 0, 2(T4), and 5 along with a trace of number 4(T3) (Figure 11). There also were traces of MIT and DIT. However it can be observed from comparison of Figures 9, 10 and 11 that compound 0, 5 and 4(T3) were less concentrated in the acid-insoluble fraction of Protamone hy- drolysate than in the butanol extract. The number and identity of iodinated compounds in thyroid proteins have already been clearly established. Thyroid pro- tein hydrolysates were chromatographed in order to ascertain if the method employed here could be used to detect and identify all known iodinated components of this gland. Figure 12 illustrates the result of the chromatography of a thyroid protein hydrolysate. It can be observed that all known iodinated compounds of the thyroid were found and identi- fied correctly. A very faint spot appeared, that could not be identified, and this is labeled with a question mark. The triangulation method for identification of unknowns by comparison to knowns, when two dimensional TLC has been employed, was discussed and illustrated in the Materials and Methods. This procedure identified compounds 2 and 4 as 50 Figure 11. Two dimensional thin layer Chromatograph of the acid—insoluble fraction of Protamone hydrolysate. Column 1: acid insoluble fraction of Protamone hydrolysate. Columns 2-5: reference compounds. 51 20 THIN LAYER CHROMATOGRAPH OF THE ACID INSOLUBLE FRACTION OF PROTAMONE HYDROLYSATE m'r‘ DI; 00 5 .® . 4 ———”-30LVENT q moms 0.MIT 0,69 4 o0 DIT 6 0.3 O, Foam: 69 2 ACID "1° 3 2 I <— u-sun /fiH‘oyanonoronu Figure 11 52 Figure 12. Two dimensional thin layer chromatograph of Thyroid Protein hydrolysate. Column 1: rat thyroid hydrolysate. Columns 2—5: reference compounds. 55 20 THIN LAYER CHROMATOGRAPH OF THYROID PROTEIN HYDROLYSATE -® 3? |- MIT ‘SOLVENT m1 5§® 0 FRONT H 0'8 T 6:”: o7? o '22:? ® ® "”0 / 4 3 2 I (—-——— N- BUTANOL/NH‘ OH /OHLOR OFORH Figure 12 54 thyroxine and triiodothyronine, respectively (Figures 9, 10, and 11). However other evidence was obtained to confirm this conclusion. The washed Protamone hydrolysate was separately diluted with 1/2 volume of 0.4 mg/ml thyroxine and triiodothyronine and then chromatographed. The chromato- gram of these two mixtures resulted in an increase of Spot density, as shown by the FFCA spray, in the areas which corresponded to T4 and T3 as identified by the triangulation method. Pauly's reagent also reacted with compounds 0, 2(T4), 5 and 4(T3) indicating the presence of a benzene ring struc- ture. The chromatographed position of the only known thyroidal iodinated thyronines, thyroxine and triiodothyronine, from thyroid hydrolysates was identical to compounds identified as thyroxine and triiodothyronine in Protamone hydrolysates. Finally, the FFCA spray obviously demonstrated that these com- pounds contained iodine. Therefore based on the evidence presented, the identity of compounds 2 and 4 was determined to be thyroxine and tri— iodothyronine, respectively. The identity of the other com- pounds was not determined; however, it can be stated that they are iodinated compounds and that numbers 0 and 5 contain a benzene ring. The only other compounds identified were MIT and DIT. Finally no evidence of free iodide could be found in Protamone hydrolysates. 55 Figure 10 demonstrates that the washed Protamone hydrolysate could be separated one dimensionally, employing the formic acid/water solvent because no contaminating sub- stances appeared when the hydrolysate was chromatographed the second time using the n-butanol/ZN NH4OH/chloroform solvent. The iodine analysis was performed according to the method previously described. The percent concentration of thyroxine and triiodothyronine, calculated on a weight basis, is pre- sented in Table 5. The thyroxine equivalent of 5.28% was determined by multiplying the percent triiodothyronine con- tent by its thyroxine potency (4.09 as determined by Reineke and Lorscheider, 1967),iresulting in a value of 2.49% and then adding the 0.79% of thyroxine to yield a final value of 5.28% thyroxine equivalent. The value of 4.09 for triiodothyronine potency versus thyroxine was used because it is in the mid— range of those published, except for the report of Stasilli et al.'(1959) which gave about twice this value. .ucoam>flsoo ocflxoumnu &mm.m mo moam> Hosam m oaoflm o» as mo Rm>.o onu msfloom cusp ocmmfimw.m mo osam> m Ga msfluHSmou Amo.¢v honouom a ocflxonmsu muH >3 m9 unmouom osu msflma DHUE >9 oosflfiuouoo mm3 ucoam>flsoo ocHxOH>£B* m 1 534333 3.» *uziomzc. m... 68H 56 .m . mz.zom>:hooo=m._. N7: MNO.H II ah. N 926255 hzmomma «15.0 mzozfloma z. mz_zom>E.ooo__E oz< wz_xom>I.—. mo mzo_._.