w . .E V . N \ _ A . ._ ..: . , . . . . , . . r fi? N 11 A E HE RIO; ~GV A N V A 1081.1 of END; . T Y 6 STATE? TH : Eree E s :EBINDI EDe AN S7 _. D' _ SERUM fbrfhe MICHIG EN. HYROXINE gigs 'BEHNE T. .. In COMPARATIVE EEWNJW; .K 1 . r * Pf. £911... £4me 2.1%“? .L._ h 1.. . .. _. firignm: .3... . in... £511" m“ - ' LZFBRJQ R Y A ‘ Michigan State F .. University ,. _. This is to certify that the thesis entitled COMPARATIVE STUDIES OF THE RELATIONSHIP BETWEEN SERUM THYROXINE AND THYROXINE-BINDING GLOBULIN presented by Kevin M. Ogon Etta has been accepted towards fulfillment of the requirements for Ph.D. ' degreein Physiology .ZKM Major professor Date May 10, 1971 0-7639 ABSTRACT COMPARATIVE STUDIES OF THE RELATIONSHIP BETWEEN SERUM THYROXINE AND THYROXINE-BINDING GLOBULIN BY Kevin M. Ogon Etta A new non—electrophoretic technique is presented for determining binding capacities of the specific thyroxine-binding globulin (TBG) in blood serum. This method is based on complete saturation of the binding sites on TBG using a mixture of radiothyroxine and unlabeled endogenous as well as exogenously added thyroxine (T4). Barbital buffer (pH = 8.6) is employed to inhibit T4 bind- ing by prealbumins. The high dilution factor of 30-35 is employed to inhibit T4 binding by albumins. Previous reports have indicated that albumins, prealbumins and a-globulins are the principal carriers of T4 in blood. Under the conditions of the present experiments, the bind- ing capacity measured is almost entirely that of TBG. Since binding capacities of carrier proteins have been reported to vary with temperature, all serum and thyroxine mixtures are incubated in a 37°C water bath in order to obtain comparable results. Kevin M. Ogon Etta Serum T4 levels are measured by a competitive binding procedure. Using the new method for measuring thyroxine binding capacity, a binding curve is documented by plotting ug T4/100 ml serum bound to TBG on the ordinate and ug total T4/100 ml serum used on the abscissa. Binding capa- cities usually rise with increasing concentrations of total thyroxine until a plateau or saturation point is reached. A point on the plateau is selected as the total thyroxine concentration to be used in determining the maximum bind- ing capacity of TBG in individual serum samples of the species in question. All determinations are made in dup- licate and these show good agreement. In preliminary experiments, 20 measurements were made of the binding capacity of TBG in a pooled sample of bovine serum. The mean value of 12.02: 0.13* Mg T4/100 ml serum obtained indicates good repeatability for the tech- nique. Serum thyroxine and binding capacities of TBG were determined in normal nonpregnant women, Holstein cows, Suffolk sheep, pigmy goats, horses and white-tailed deer. Serum T4,ug % The ratio of in each TBG binding capacity, T4 pg % species is termed Saturation Index (SI). The ungulates studied, except deer, have a mean Saturation Index of 0.73 i 0.03 and this was not significantly different from *Standard error of the mean. Kevin M. Ogon Etta the index in women. Saturation Indexes are also unaltered in pregnant cows, sheep and in women taking oral contra— ceptive pills. All these species have in common the fact that their TBG is the major carrier of T4. In deer, where albumins have been reported to transport quantitatively more thyroxine than globulins, the Saturation Index is significantly higher than that of other higher mammals studied. Deer also have significantly higher serum thyroxine levels than any other higher mammal studied. Since thyroxine—binding capacities of deer TBG is comparable to that of the other mammals, the higher Saturation Index indicates T binding to proteins other 4 than TBG. In bovine fetuses, there is a gradual increase of serum T4 and TBG capacity during the second and third trimesters of pregnancy. Quantitatively, the two para- meters are almost identical and yield Saturation Index values of 1.05 and 1.13 during the second and third tri— mesters, respectively. After birth, when TBG levels rapidly decline, the excess thyroxine is released from protein binding thus becoming available for metabolic uses. It is suggested that this reserve of quickly avail- able extrathyroidal T4 plays a vital role in the adjust- ment of the newborn calf to its environment. Serum thyroxine levels of thyroprotein—fed cows are significantly elevated over those of untreated cows but with no corresponding change in the thyroxine-binding Kevin M. Ogon Etta capacity of TBG. This results in a significantly higher Saturation Index in the thyroprotein-treated than in the untreated cows. The mean Saturation Index of 0.97 i 0.04 in treated animals also indicates that TBG is the major T4 carrier protein in cows and any T4 in excess of the binding capacity of TBG is rapidly cleared. Another physiological state during which the Saturation Index of cows is substantially altered is lactation. Here serum thyroxine levels are significantly depressed, probably because of the high intensity of lac- tation and consequent intense competition for iodine between the thyroid and mammary glands. Binding capacities of TBG are, however, unaltered and a significant depression of Saturation Index results. In nonlactating pregnant cows, sheep and rats, serum T4 and Saturation Index values are statistically no different from the values in open animals. Open rats have serum T4 levels of 6.20 i 0.17 ug/lOO ml serum. Since the binding capacity of their TBG is only 1.73 i 0.23 ug T4/100 m1 serum, Saturation Index is 4.06 i 0.41. Guinea pigs and birds show negligible T4-binding TBG capacities and relatively low serum thyroxine levels. Male chickens have higher T levels than those of 4 nonlaying chickens whose T levels are higher than those 4 of the layers. Male turkeys are different from other birds studied in having TBG T -binding capacities of 4 Kevin M. Ogon Etta 1.34 i 0.24 ug T4/100 m1 serum, a value which is statis- tically higher than zero. COMPARATIVE STUDIES OF THE RELATIONSHIP BETWEEN SERUM THYROXINE AND THYROXINE-BINDING GLOBULIN BY Kevin M. Ogon Etta A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1971 DEDI CAT I ON This thesis is dedicated to the memory of my late grandmother, Lucy Baarong Obi, in appreciation of the shining example her life was to all her children and grandchildren. It is also dedicated to my—- mother, Maria-celine Okaja Odu, wife, Maria Ndik Etta and guardians, Joseph Obi Etta and Hon. Michael Etta Ogon for contributing in countless vital ways to the successful completion of my entire educational program. ii ACKNOWLEDGEMENTS The writer feels deeply indebted to Professor E. P. Reineke for his wise counsel in the planning and execution of this study. His patient cooperation and sympathetic understanding have been an invaluable stimulus toward the successful completion of the author's program at Michigan State University. Special thanks are due to Professor R. K. Ringer for the supply of experimental bird sera, advice in the interpretation of avian data and constant personal encour— agement in the course of this study. The writer is also grateful to Dr. Alvin E. Lewis and Mrs. Margaret L. Shick for the supply of human blood samples, Dr. A. J. Pals for guinea pig samples, Dr. W. J. Youatt for deer samples and Mr. Russel Erickson for the blood samples from thyroprotein—treated cows. Special gratitude is due to Dr. Walter Wan for permission to use the thyroxine values of horse serum reported in his Ph.D. thesis, Dr. F. L. Lorscheider for the thyroxine values of sheep and lactating cows reported in his Ph.D. thesis and to M. V. Hernandez, D.V.M., for the thyroxine values of iii bovine, fetal, neonatal and pregnant heifer sera that appeared in his M.S. thesis. Sincere appreciation is due to the Agricultural Experiment Station, Michigan State University, who provided the funds that made this study possible and to the Agency for International Development for initial financial support. iv TABLE OF CONTENTS Page DEDICATION . . . . . . . . . . . . . . . ii ACKNOWLEDGEMENTS . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . .viii INT RODUCT ION o o o o o o o o o o o o o 0‘ o 1 REVIEW OF LITERATURE . . . . . . . . . . . . 4 Thyroxine-Binding Proteins . . . . . . . 4 TBP During Pregnancy . . . . . . . . . 5 Effects of Lactation . . . . . . . . . 7 Binding Protein-Thyroid Hormone Relationships During Fetal Life in Newborn and in Maternal Serum . . . . . 8 Effects of pH and Buffer on T4-Binding Capacities of TBG . . . . . . . . . . 12 Effect of Temperature . . . . . . . . . 13 Effect of Dilution . . . . . . . . . . 14 Sexual Maturity and Estrogen Secretion . . . l4 Techniques for Measuring Thyroxine- Binding Capacities . . . . . . . . . l7 STATEMENT OF THE PROBLEM . . . . . . . . . . . 23 MATERIALS AND METHODS . . . . . . . . . . . . 24 Chemicals . . . . . . . . . . . . . 24 Serum Samples . . . . . . . . . . . . 25 Apparatus . . . . . . . . . . . . . 26 RESULTS GENERAL Repeatability . . . . . . . Thyroxine-Binding Globulin T4- Binding Curves . . . . . . . Thyroid Parameters of Man and Other Species . . . . . . . . . . Effect of Pregnancy on Serum T4 and TBG Capacities . . . . . . . Effect of oral Contraceptives on Serum and TBG Capacities . . . . . Satgration Index in Thyroprotein- Treated Cows . . . . Thyroxine- Binding Capacities of TBG During Lactation . . . . . . Thyroxine- Binding Capacities of TBG in Bovine Fetus and Newborn Calves . . Avian Thyroid Parameters . . . . . DISCUSSION . . . . . . . . . . APPRAISAL OF NEW TBG-SATURATION TECHNIQUE . . SUMMARY APPENDICES A. B. C. D. Thyroid Parameters of Sheep During Various Physiological Conditions . . . . E. Thyroid Parameters of Newborn Calves from the lst to the 6th Day of Birth . . . F. Repeatability Studies: TBG T4-Binding Capacities and Saturation Indexes of Serum Samples from One Heifer . . . . G. Thyroid Parameters of Rodents . . . . H. Avian Thyroid Parameters . . . . . . I. Saturation Technique for Measuring Binding Capacity of TBG . . . . . . . . REFERENCES . . . . . . . . . . . Thyroid Parameters of Deer, Pigmy Goats and Horses . . . . . . . . Thyroid Parameters of Cows in Various Physiological Conditions . . . . Bovine Fetal Thyroid Parameters During the Second and Third Trimesters of Pregnancy vi Page 28 28 28 30 33 33 37 39 41 43 46 61 66 69 71 74 76 78 82 83 85 88 99 LIST OF TABLES Table . Page 1. Thyroid Parameters of Women and Various Species of Animals . . . . . . . . . 31 2. Mean (1 Std. Error) Thyroidal Parameters in Open, Lactating and Pregnant Sheep and cows 0 O O O O O O O O O C O O O 34 3. Mean (1 Std. Errors) of Thyroidal Parameters of Open and Pregnant Rats and Guinea Pigs , , 35 4. Thyroxine-Binding Capacities of TBG and Serum Thyroxine Levels (ug T4/100 ml Serum) in Control Women and Those Taking Oral Contraceptives . . . . . . . 36 5. TBG T4-Binding Capacities, Serum T4 (ug T4/100 ml Serum) of Pregnant Thyroprotein-Treated and Untreated Cows. II Saturation Indexes of Both Groups . . . . . . . . . . . . 38 6. Mean TBG Saturation Indexes (1 Std. Errors) of Fetus, Mother and Neonatal Calves . . . 42 7. Mean (1 Std. Errors) of Thyroidal Parameters of Chickens and Turkeys . . . . . . . . 44‘ vii LI ST OF FIGURES Figure Page 1. T ~binding Curves of TBG in Women and Three Species of Animals . . . . . . . 29 2. Thyroidal Parameters of Bovine Fetus, Neonate and Mother. (Serum T4 Values by Hernandez, 1971; Heat Production Data by Roy et al., 1957) . . . . . . . 40 viii INTRODUCTION The thyroid gland, through its hormones, affects the-integrity and function of every major system in the bodies of higher animals. It is not only involved in basal metabolism and temperature regulation, but also in reproduction and lactation, cerebration and development, and even in mineral metabolism. Like all other hormones, thyroid hormones complement the nervous system in main- taining an effective communication and coordination between the various organs and tissues. An accurate assessment of the status of the thyroid in different physiological con- ditions is, therefore, of the utmost importance. Over the years, thyroidal uptake of iodine, thyroid secretion rates, total serum thyroxine, thyroxine degradation rates, protein bound iodine (PBI) and meta- bolic rate have all been studied in an attempt to estab- lish reliable indices of thyroid function in man and various animals. ,In comparatively recent years, it has become apparent that a parameter of possibly equal import- ance is the thyroxine-binding capacity of thyroxine— binding proteins. _ Since 1952, it has been known that human serum thyroxine has a maximum affinity for d-globulins, an intermediate affinity for prealbumins and only feeble affinity for albumins. Extensive studies have been under- taken to determine alterations of thyroid status during different physiological states in man. The data during one such state, pregnancy, indicated augmented thyroidal avidity for iodine, thyromegaly and an increase in the circulating levels of thyroid hormone. Although this triad of anatomical and functional alterations is ordinarily considered to be diagnostic of thyrotoxicosis, it was associated in early pregnancy at least, with an unequivo- cally euthyroid state. It was postulated that alterations in the interaction of thyroid hormone with serum proteins might provide a clue to these paradoxical findings. Since early reports indicated that thyroid hormone is largely bound to a specific u-globulin in human serum, attention has been directed to this protein in most of the recent investigations. Most investigators up to now have used various electrOphoretic techniques in studying the capacity of the Specific d—globulin to bind thyroxine. A comparison of the reported electrOphoretic patterns of various animal sera shows that these patterns vary widely both in regard to the number of distinct components and with respect to the relative proportion of each component. Such variations are attributable, at least in part, to heavy trailing and other drawbacks of electrophoresis that have been pointed out in recent reviews. Even the techni- que of reverse—electrophoresis that was introduced to obviate trailing still has several other drawbacks, not the least being its unsuitability for routine application. In the present study, a new non-electrophoretic technique for measuring the binding capacity of TBG is presented. This technique is suitable for routine use, avoids the drawbacks of electrophoresis and employs a resin-impregnated sponge for separating protein-bound from unbound thyroxine. The binding of thyroid hormone in human serum has been extensively studied. There have, however, been fewer reports of thyroxine-protein interactions in other species, even though currently available information suggests important species differences in the thyroxine—binding proteins. Experiments to be reported in this study were planned to obtain data on the relationship between plasma level of T and TBG in several different species and to 4 explore the possibility of changes within species during varying physiological states. A new thyroidal parameter, the Saturation Index, is introduced to express the relationship between serum T4 levels and the binding capacity of TBG. REVIEW OF LITERATURE Most of the early findings in the field of physiology were the results of investigations undertaken to improve the practice of medicine, understand human diseases, and cure them. Findings concerning thyroxine— binding proteins were no exception. Thyroxine—Binding Proteins The existence of a Specific thyroxine-binding pro- tein (TBP) with a very high affinity for thyroxine (T4) was first convincingly demonstrated in 1952 by Gordon and his co-workers. When human serum containing 131I-T4 was subjected to zone electrophoresis on paper, the binding protein exhibited the properties of an d—globulin at pH 8.6. This protein was designated thyroxine—binding d-globulin (TBG). Subsequent to the discovery of TBG, the existence of a second important thyroxine—binding protein in human serum, thyroxine-binding prealbumin (TBPA), was demonstrated (1958). A third binding protein in human serum, albumin, appears to be of less importance physiolo- gically than the other two carriers of the hormone. Thyroxine affinity for globulin is much stronger than that for prealbumin and even stronger than the affinity for albumin. Thyroid hormones secreted into the blood from the thyroid gland are bound by thyroxine—binding proteins. In mammals, the affinity of thyroxine for the binding pro- teins is about 3-4 times that of tri—iodothyronine. Evi— dence has been adduced to show that tri-iodothyronine is bound either feebly or not at all to TBG in 3122 (Zanino- vich §£_§l., 1966; Robbins and Rall, 1957). Most of the review will, therefore, center around the binding of thyroxine by the binding proteins. For thyroxine to participate in tissue metabolism, it has to be freed from its binding proteins. Consider— able interest was, therefore, generated in the physiolo— gical role and physicochemical properties of the specific thyroxine—binding proteins. Many researchers have linked the variations of thyroxine—binding proteins in various physiological conditions with an alteration in the level of thyroxine in serum. TBP During Pregnancy Dowling et_al. (1956b) reported a marked increase in TBP in human serum during pregnancy. It has also been observed that the thyroid glands of pregnant women have an augmented avidity for iodine (Pochin, 1952). Circulating levels of thyroxine are believed to rise during pregnancy (Heinemann §E_al., 1948; Russel, 1954). These three func- tional alterations are ordinarily considered to be diagnos- tic of thyrotoxicosis. Paradoxically, however, the above findings are neither accompanied by the symptomatic stigma of hyperthyroidism, nor are they associated, at least dur- ing the first half of pregnancy, with an increase in the basal metabolic rate. The increased thyroxine-binding protein of pregnancy may thus be directly related to both increased avidity for iodine by the thyroid and increased circulating levels of thyroxine. Precise cause and effect relationships between these parameters are still difficult to establish. The thyroid gland would need to take up more iodine at a faster rate to keep up with the increased synthesis of thyroxine and increased circulating levels in blood serum. More iodothyronines are therefore synthe- sized and secreted from the thyroid gland. Whether this increased level of thyroid hormone synthesis and produc— tion is the result or the forerunner of increased levels of thyroid hormone-binding proteins is a subject of intense debate and research. Many researchers prefer to merely implicate increased thyroxine-binding protein with increased circulating thyroxine levels (Dowling §t_al., 1956a). Others suggest that the increased circulating levels of thyroxine are a consequence of increased serum binding proteins (Tepperman, 1962). According to the latter View, a small amount of free thyroxine in the blood is necessary for the feedback mechanism between the thyroid gland and the production of thyrotropic hormone by the adenohypophysis. If this free thyroxine level is decreased by an increase in the binding proteins, more tropic hormone is put out from the adenohypophysis. This stimulates thyroid gland activity and more thyroxine is synthesized and secreted to bring the unbound thyroxine level in the blood to a normal value for the species. The concept of a relationship between the serum thyroxine level and bind- ing capacity of TBG might also be the key to the levels of T4 and TBG capacity for T4 during lactation, in fetal blood, in newborns and in near-term and postpartum animals. Effects of Lactation Thyroid secretion rates in rats have been shown to fall from 2.58 to 0.99 ug T4/100 gm body weight/day during lactation (Lorscheider, 1970). At the same time serum T4 declined from 3.94 to 1.96 ug T4/100 ml. So both methods of thyroid evaluation show marked and significant reductions of more than 50% in lactating rats. Serum thyroxine levels have also been shown to drop significantly during peak lactation in ewes and in cows (Lorscheider, 1970). Flamboe and Reineke (1959) have reported a lower thyroid secretion rates in lactating goats compared to the nonlactating ones. In 1961, Iino and Greer reported marked decrease in the thyroidal uptake of radioiodine during lactation. Piecing together these several reports, a consistent picture emerges. During lactation, the thyroid takes up less iodine, and so synthesizes less thyroid hormone. Thyroid secretion rate is consequently lower. This in turn results in a low serum thyroxine level. The low radioiodine uptake by thyroid glands dur- ing lactation appears to be related to the successful competition of mammary glands for iodine during this time. Flamboe and Reineke (1959) reported that considerable iodide is secreted in milk. It would be of interest to find out the picture of T4—binding capacities of thyroxine- binding protein as the thyroid activity changes during lactation. So far as this writer is aware, there are no reports in the literature on this subject. Fell et_§l. (1968) reported a striking rise in y-globulins of blood serum during lactation in the ewe. However, y-globulins are more important as immune proteins than as carriers of thyroid hormones. If the binding protein-thyroxine relationship during pregnancy is any guide, there should be a corresponding relationship between serum T levels and the binding proteins during 4 lactation. Binding Protein-Thyroid Hormone Relationships During Fetal Life in Newborn and in Maternal Serum Thyroid follicles begin to appear in the human fetus between the 7th and the 12th week of pregnancy (French and Van Wyk, 1964; Chapman gt_al., 1948). In the bovine, measurable amounts of iodine were first detected in fetal thyroids at 60 days of age (Wolff et al., 1949). A direct relationship appears to exist between calf fetal thyroid iodine and each of the three growth parameters-~body weight, crown-rump length and calculated age (Nichols et_al., 1949; Wolff gt_al., 1949). Wolff and his co-workers also reported a progressive increase in the iodine con- centrating capacity of fetal thyroid tissue with age. Gorbman gt_al. (1952) sacrificed two pregnant cows 24 hours after radioiodine injection and found that fetal thyroids 1311 as the maternal thyroids. contained twice as much Fetal thyroglobulin was also found to be 27 times more radioactive than in the mothers, more than one—fifth of the radioactivity in fetal thyroids being ascribed to freshly synthesized thyroxine. In sheep, with an average gestation period of 150 days, the fetal thyroids start to accumulate iodine by the 50th day of gestation and formu- lation of the thyroid follicles is observed histologically on the 42nd day (BarneS‘gE_al., 1957). Rats (gestation period 21-22 days) acquire the functional ability to store iodine around the l8th-l9th day of gestation. This abil- ity may be correlated with the first differentiation of follicles complete with lumen. At 21 days of gestation the rat is able to synthesize mono-and di-iodotyrosine as well as tri— and tetra-iodothyronine. For most of the species discussed above, therefore, fetal thyroids start functioning quite early during gestation. In humans, sheep, cows and goats, fetal thyroids are already metab- olizing iodine by the end of the first trimester of 10 pregnancy. The binding capacities of the thyroid hormone carrier proteins can be expected to play a role in regu- lating observed levels of thyroid activity in the fetus. Russel gt_al. (1964) reported a mean fetal thyroxine- binding globulin T4-binding capacity of 29.1 pg T4/100 ml of serum as compared to a maternal capacity of 42.1 ug T4/100 ml serum in humans. Dowling gt_al.(l956b) reported that the capacity of TBG for T in fetal humans was 1.33 4 times greater than the level in nonpregnant adults but still about 0.5 times the level in their mothers. There seems to be agreement that pregnant women have much higher TBG capacities for T4 than the binding capacities of the fetal TBG. This raises several questions. Is the placental barrier which separates the fetal from the maternal circula— tion permeable to the thyroxine-binding proteins? Is the lower binding capacity for T4 in fetal blood serum a reflection of a higher saturation and so a higher level of serum thyroxine? Several workers have reported that placentae in many animals are permeable in varying degrees to thyroxine (Osorio and Myant, 1962; Hoskins gt_§l., 1958; Reineke and Turner, 1941; Zondek, 1940; Contopoulos gt_al., 1964; Monroe et_al., 1951). An increase in the transplacental passage of thyroid hormones as human pregnancy advances has also been demonstrated by Osorio and Myant (1962). This increase has been attributed to either an increase in the permeability of the placenta, a decrease in the 11 thickness of the membranes which separate fetal from maternal circulations or an increase in placental blood flow. The most important factor controlling the transfer of T4 across placentae seems to be the difference in com- position, affinity and binding capacity of the serum T4- binding proteins between mother and fetus. The higher the thyroid hormone gradient, the greater the transfer across the placenta within the limits of the placental permea- bility. From reports by Myant (1964) and Robin gt_al. (1969), the slightly higher concentration of free thyroxine in the fetal blood results in a positive net diffusion from fetus to mother, at least in man. Higher levels of free thyroxine in human fetal serum reflect the lower binding capacity by the thyroxine- binding proteins that has been reported by Russel gt_al. (1964). So the same kinds of binding protein-thyroxine interrelationships which have been observed in pregnancy and during lactation may also obtain in fetal, neonatal and postnatal animals. For instance, there have been several reports of very marked increases in the serum thyroxine levels of human babies within the first 30 minutes after birth (Fisher et_al., 1964a; Fisher and Odell, 1969; Robin §£;§l., 1969). Fisher and his co- workers suggest that cold exposure of the newborn is responsible for the initial thyroid hyperactivity. It would be of interest to find out what changes, if any, occur in the thyroxine—binding proteins during gestation and soon after birth. 12 The View of a close relationship between the serum thyroxine-binding proteins and the circulating level of thyroxine brings into focus an important question. Is there really an absolute increase in the thyroxine-binding globulin? Or is there rather an increase in those molecu- lar or chemical characteristics of the proteins which determine their affinity for thyroxine? To date, there is no conclusive answer, but some light has been shed on this question by studies of factors which influence the affinity of thyroxine-binding proteins for thyroxine. The following factors have been known to influence this affinity: pH and buffers Temperature Dilution Sexual maturity Estrogen Effects of pH and Buffer on T4-Binding Capacities of TBG The effect of pH on the capacity of serum proteins to bind thyroxine has been studied by Robbins and Rall (1960), Keane gt_al. (1969), and Lutz and Gregerman (1969). At pH 8.6, using barbital buffer, the binding of thyroxine to TBG is unaffected, but binding to prealbumin is markedly reduced (Keane gt_al., 1969; Robbins and Rall, 1960). Lutz and Gregerman (1969) used sodium phosphate buffer solution prepared by mixing ratios of mono- and 13 disodium salts. They found that the binding of thyroxine to albumin was maximal at pH 8.6 using sodium phosphate buffers. At pH values below and above 8.6, the binding to albumin was markedly reduced (Antoniades, 1960). Robbins and Rall (1960) in a very extensive review sug- gested that barbital buffer competed with thyroxine for the binding sites on the prealbumin. On the basis of electrophoretic studies using agar gels of physiologic pH (7.4), Hollander and co-workers (1962), estimated that the normal distribution of hormone between the three carriers is approximately 60 per cent with TBG, 30 per cent with TBPA and 10 per cent with albumin. Barbital buffer seems to be unique in competitively preventing the binding of thyroxine to prealbumins. Effect of Temperature Temperature importantly affects the binding of thyroxine to its carrier proteins. Perhaps the strongeSt evidence of this effect was provided by the work of Murphy and Pattee (1964). Standard curves for the binding of thyroxine by its carrier proteins were determined after equilibrating serum samples at various temperatures. Results indicated that binding was increased at lower temperatures. Thyroxine-binding capacity at 4°C is higher than at 23°C. Binding is lower at 30°C and even less at 40°C. For binding capacities to be comparable, therefore, they must be determined at similar temperatures. 14 Effect of Dilution Besides temperature effects, Murphy and Pattee (1964), as well as Keane gt_al. (1969) have shown that dilution of either plasma or serum decreases the normally feeble binding of thyroxine to albumin. The higher the dilution, the less thyroxine is bound. A decrease of the weak binding to albumin meant that almost all binding could be attributed to TBG when _using barbital buffer at pH 8.6. This finding offered a subtle method for determining the thyroxine-binding capacity of TBG. Murphy and Pattee (1964) found that the binding capacity at 1:12th dilution was much more than double that at 1:32nd dilution. If determinations of binding capacity were made at pH 8.6 using barbital buffer and at about a 1:32 dilution, this would be a measure of the binding capacity of almost entirely TBG. The limit- ing factor here has to be the lower limit of serum dilu- tion at which the thyroxine-binding globulin capacity can still be measured. Sexual Maturity and Estrogen Secretion Another factor which determines the binding capa- city of thyroxine carrier proteins is the maturity of the animal. In a study of thyroxine-binding to the serum protein of adolescents and children, Riecansky (1967) demonstrated the importance of puberty. Thyroxine-binding to TBG in prepuberal children was shown to be unaffected 15 by either sex or age. Binding to prealbumin was not dif- ferent in boys or girls at the age of ten. In 15-year- old subjects, however, the girls showed a higher binding capacity in their serum TBPA than the boys. Riecansky suggested that these differences were probably due to a higher level of sex hormone secretions in girls of this age. The greater ability of TBPA to bind thyroxine in these circumstances has also been shown by Ingbar (1963). Other workers have demonstrated more specifically, the link between sex hormones and thyroxine-binding pro- teins. Alterations in TBG capacity for thyroxine have been repeatedly demonstrated for serum of gravid women. In an attempt to explain the origin of such alterations, several researchers suggested that the profound changes in the metabolism of estrogen during pregnancy might at least be contributory. During both the pre-partum and post-partum periods, changes in the level of serum PBI and thyroxine-binding are qualitatively similar and temporarily coincident (Dowling et_al., 1965b). It has also been demonstrated that large doses of estrogen when administered to males or to nonpregnant females induced increases in the serum PBI level comparable to those which occur during normal pregnancy (Engstrom et_al., 1952). Finally, a profound and progressive increase in the elaboration of estrogen is a concommitant of normal preg- nancy (Sunderman and Boerner, 1949). It therefore seemed logical to study the effects of estrogen on the 16 thyroxine-binding capacity of TBP in various subjects. Such a study was undertaken by Dowling and his co-workers (1956a). All the human subjects received diethylstilbes— terol in daily oral doses of 30 mg for 5 weeks and 60 mg after that. One patient with treated myxedema received 30 mg daily for four weeks and subsequently, 60 mg daily. Also included in this study was a patient with panhypo- pituitarism. Measurement of thyroidal accumulation of 1311, BMR, serum PBI level and thyroxine-binding capacity of TBP were made at intervals prior to, during and follow- ing the administration of estrogen. In all eumetabolic patients, the administration of diethylstilbesterol was associated with an increase in the percentage of added thyroxine bound to TBG. This increase was accompanied in all such patients with the previously observed increase in PBI levels and these alterations were comparable to those occurring during normal pregnancy. These effects of diethylstilbesterol were not dependent on the normal functioning of the thyropituitary axis, since they were noted in patients with hypopituitarism and with treated primary myxedema. The parallel between pregnancy and the administration of synthetic estrogens is thus very close. This parallel has also been borne out by the work of Musa §E_al. (1969). Dowling and his group also observed an increase in the capacity of TBG for thyroxine during estrogen administration. These findings suggest that the marked augmentation of thyroxine—binding by TBG which 17 occurs during human pregnancy may, at least in part, result from the influence of endogenous estrogen. Techniques for Measuring Thyroxine— Binding Capacities From the foregoing, it is apparent that a measure of TBG capacity is an important tool for evaluating not only thyroid economy but also sexual maturity and thyroidal changes during pregnancy. Almost as soon as Gordon §E_al. (1952) characterized the three thyroxine—binding proteins, modifications of existing electrophoretic methods were employed to determine the capacity of each of the binding proteins for thyroxine. One of the first findings of such early studies is the dependence of the results on the method of determination. Methods range from dialysis to electrophoresis and, lately, immunoadsorption. Perhaps the most widely used and most variously modified is electrophoresis. An outline of this technique is presented here as the basis of a review of other techniques. As employed by Robbins (1956), electrophoresis involved passing a constant electric potential of 100 volts for 24 hours through strips of Whatman number 3MM filter paper. Barbital buffer, pH 8.6 and ionic strength 0.1 was used. Each strip of paper was 3.75 cm wide and was sus- pended horizontally in a closed system between glass plates by means of taut silk threads along each edge. The ends of the paper strips dipped into two vessels, each 18 containing 1325 m1 of buffer. The strips were moistened with buffer and placed in the chamber approximately 30 minutes before the serum was added. Glass plates, which were 3.4 cm apart, were lined with moistened filter paper. In a conventional run, Robbins (1956) applied 30 ul of a serum 131I—L—T4 mixture in a band near the cathodal end of the strip, 9 cm from the fluid level. The equalizing tube between buffer vessels was then closed and a constant electric potential applied for 24 hours. After the strips were dried, radioactivity was measured with a Geiger Muller counter. The proteins in the strips were then stained with bromphenol blue and quantified. Tiselius and Flodin (1953) reported that in con- ventional electrophoresis, adsorption of migrating sub— stances on the supporting medium frequently occurred. This adsorption was probably why Robbins (1956) could not get a saturation point for TBG--when he flooded-—human serum with exogenous thyroxine. Robbins then introduced reverse electrophoresis as one method of obviating the difficulty of adsorption on the supporting medium. Reverse electrophoresis could presumably eliminate artifacts due to the adsorption of albumin—bound thyroxine on the filter paper medium since the serum globulin would no more migrate in the path of albumin. The movement of albumin toward the anode was just balanced by a flow of buffer in the opposite direction. This resulted in a displacement of globulins toward the cathode. But reverse—flow 19 electrophoresis itself produced more diffusion and some distortion of the protein bands. All electrophoretic media currently used for the study of serum thyroxine-binding proteins present inherent disadvantages (Launay, 1966). Gordon and his co-workers as well as the other investigators who first discovered the existence of a specific thyroxine—binding protein in human serum used paper, the adsorptive properties of which produced heavy trailing and poor resolution of the protein bands. These bands were very clearly separated in starch—gel (Rich and Bearn, 1958), and starch block (Larson et_al., 1952). The preparation of these media was, however, delicate and time consuming. Localization and quantification of radioactive fractions were also difficult and imprecise. Electrophoresis on slides coated with agar gel (Digiulio §E_§l., 1964) presents the same drawbacks. The great variation in the results of electro- phoretic methods was perhaps best illustrated in a review of these methods by Woeber and Ingbar (1968). They pointed out that of an endogenous concentration of T4 about 30 per cent migrated with TBPA in agar gel at pH 7.4 (Hollander §t_al., 1962), 30-45 per cent migrated with TBPA in filter paper at pH 8.6 (Ingbar and Freinkel, 1960), while in starch gel values which vary from 10-60 per cent have been reported (Blumberg and Robbins, 1960). Electrophoretic methods which have commonly been employed to assess the apportionment of endogenous thyroxine 20 among the binding proteins inevitably raised the possibility that artifacts were produced by the pH, the supporting media or buffers used. Artifacts could also be produced during the separation of proteins from one another or by the electrical field itself. It is not surprising there- fore that the properties of endogenous T4 associated with TBPA as judged from electrophoretic analyses have varied greatly. Disc electrophoresis has proved unsuitable for the study of binding proteins because a significant proportion of the radioactivity of 131 I—labeled thyroxine was lost in the sample gel. Besides, most of the radiothyroxine migrated far ahead of any protein fraction (Launay, 1966). Cellulose acetate membranes seemed more promising. Since they were introduced by Kohn (1957) their use for the study of thyroxine-carrying proteins has been suggested by Tata et_al. (1961) and by many others. Cellulose ace— tate appears to be a medium ideally suited for this pur- pose, since it is ready to use, can be handled and seems like paper. It gives fast separation and high resolution like gel media and is reported as not adsorbing protein, thereby eliminating trailing. Initial experiments using cellulose acetate, however, revealed the presence of a thyroxine-binding component cathodal to all three thyroxine-binding proteins so far recognized. Further investigation showed this component to be due to protein — r 2 1 trailing. This would substantially affect the determina- tion of the capacities of the thyroxine—binding proteins. Recently Woeber and Ingbar (1968) presented an immunoadsorption technique. This employed a rabbit anti- serum specific for human serum and designed to remove thyroxine-binding prealbumin from serum completely without affecting the T4-binding activity of the TBG. Only about 15 per cent of the endogenous thyroxine was bound to TBPA judged from results by this method. But this method, like the electrophoretic methods, is unsuitable for routine work. Some method that is reliable and obviates the drawbacks of electrophoresis while being suitable for routine work would be an invaluable tool for research in this area. Such a method was suggested by the work of Murphy gt_al. (1963) on corticosteroid—binding globulin (CBG) . The relationship between thyroxine and thyroxine— binding globulin is in many respects similar to that between cortisol and corticosteroid—binding globulin. When methods based on the principle of protein—bound isotopic competition were developed by Murphy and her group for cortisol and other steroids in plasma, this similarity prompted the investigation of the application of the same principle for the determination of plasma thyroxine. In 1969, Keane §E_al. modified Murphy and Pattee's technique to allow the study of plasma protein— thyroxine interactions. 22 Very few comparative studies on T —binding proteins 4 in diverse species have been made. An investigation by Tanabe gt_§l. (1969) was perhaps the most extensive of these. They employed radioautography after cellulose acetate electrophoresis. This technique may have a few of the drawbacks of electrophoresis and may not be suit— able for routine work but seems to be quite reliable. The thyroxine-binding capacity of d—globulin was found, by this technique, to be high in ungulates as compared with other mammalian orders and the lower vertebrates. In horses and dogs most radioactivity was found in albumin and d—globulin. No apparent thyroxine-binding a-globulin was detectable in Rodentia. Bound radioactivity was found only in plasma albumin in guinea pigs. In rats most radioactivity was bound to albumin and very little to post-albumin. In lower vertebrates such as Aves, Reptilia, Amphibia and Pisces, no thyroxine-binding a-globulin was found. Radio-thyroxine concentrated in plasma albumin in some species (chicken, duck, fish) but in other species (pigeon, snakes, lizards, frogs) the radio—thyroxine con- centrated both in albumin and prealbumin. The study cited above showed that specific thyroxine-binding d-globulin occurred only in mammals. All other vertebrate classes including birds had proteins that were capable of binding thyroxine less tenaciously (Farer §E_al., 1962b; Tanabe et all, 1969). STATEMENT OF PROBLEM In the research to be reported endogenous T4 in each serum sample is first determined by the Tetrasorb- 125 method.* Then a measurement of the total T -binding 4 capacity of TBG is made by the new method devised in our 131 laboratory. In this method cold T4 and I—labeled T 4 are added in excess to completely saturate the T4-binding sites on TBG. The excess or unbound T4 is absorbed by a resin-impregnated sponge. From the difference in radioactivity counts before and after this separation and the specific activity of the total T in the liquid 4 mixture the binding capacity can be expressed in terms of T bound per 100 ml of serum. Barbital buffer at pH 4 8.6 is employed to competitively inhibit binding of T4 on prealbumin (Robbins and Rall, 1960), and high dilution of the serum is used to minimize binding to albumin (Tata and Shellabarger, 1959). Comparative data on serum T and T4-binding capa— 4 city of several species of mammals and birds are presented together with data on physiological variations observed in serum of women, cattle, sheep, rats and guinea pigs. *Abbott Radiopharmaceuticals, North Chicago, Illinois. 23 MATERIALS AND METHODS Chemicals Resin—impregnated polyurethane sponges were donated by Abbott Radio—Pharmaceuticals, North Chicago, Illinois. When stored at 4°C these sponges remained reliable media for separating free thyroxine from pro- tein-bound thyroxine for several months. A primary standard, crystalline, free thyroxine was purified by Professor E. P. Reineke from monosodium thyroxine penta- hydrate. The free thyroxine showed only a single compo— nent when checked by thin—layer chromatography. A standard aqueous stock solution was prepared using this purified thyroxine to give a concentration of 5 pg T5 per ml. 131I—L-T4 was purchased from Abbott Radio— Pharmaceuticals. Working solutions of various concentra- tions were prepared from both the stock T4 and the labeled T4 solutions as described in detail in Appendix I-—4 and 5. All solutions were stored at 4°C. At this temperature the solutions remained stable and usable for upwards of 2-3 months. All containers used for storing stock and working standards were siliconized. This greatly reduces the adsorption of thyroxine to glass. 24 25 Serum Samples Most of the samples used in this study were obtained from the Michigan State University Farms and Small Animals Laboratory. Blood samples were generally allowed to stand for 4-6 hours at room temperature before being centrifuged for at least 20 minutes at 10009. Avian blood samples which yielded relatively less serum than others were spun for as long as 60 minutes. Serum obtained from the blood samples was frozen and stored for periods of several months. Before use, the serum samples were thawed and, along with thyroxine solutions, were brought to room temperature. Blood samples of female University students and workers, taking oral contraceptive pills and those not taking them, were obtained from Olin Medical Center, Michigan State University, East Lansing. In the study of the effects of thyroprotein treat— ment on thyroid parameters of cows, blood samples were taken from pregnant cows on the day before the initiation of thyroprotein treatment. Ten grams/day of thyroprotein were then orally administered to each cow for 6 days. Blood samples were again obtained from the cows the day after the last daily dosage of thyroprotein. Cows #840 and 841 were 3 to 4 years old and the rest were first- bred heifers. Deer blood samples were obtained from white-tailed deer (Odocoileus virginianus) in captivity. These were 26 being fed a well balanced diet but otherwise retained their wild instincts. All cows used were Holsteins and the sheep were Suffolks. Apparatus Gamma radiation of the 131 I-T4 was counted in a scintillation counter (Nuclear Measurements Corporation). Thorough mixing was achieved in about 30 seconds using a Vortex Deluxe Mixer (Scientific Products Division of the American Hospital Supply Corporation, Evanston, Illinois). Since the reliability of the results of this technique depends very much on the precision of the small volumes measured, a Syringe Microburet (Micro-Metric Instrument Company, Cleveland, Ohio) was used for all volume measure- ments except the barbital buffer. Each syringe used in these measurements was calibrated to determine the equivalence of one division of the micrometer by weighing the water delivered using that syringe. In this way, all volumes were measured to the nearest 0.5 ul. Serum thyroxine levels were determined by the Tetrasorb-125 resin—sponge technique as modified by Hernandez (1971). A curve for the binding capacity of TBG at varying thyroxine concentrations was documented for each animal species. The concentration of thyroxine required to saturate the thyroxine-binding sites of TBG at 37°C was selected from the plateau of the binding curve and this 27 concentration was used to determine the binding capacities of the TBG of several individuals of the particular species. Determinations of T4—binding capacities were, in each case, run in duplicate according to the sequence detailed in Appendix I—7. RESULTS Repeatability In the initial experiments, a binding curve was established for a pool of steer serum. Twenty replica- tions of the TBG T4—binding capacity of this serum yielded a mean value of 12.02 i 0.13* ug T4/100 m1 serum (Appendix F). Thyroxine~Binding Globulin T4-Binding Curves Thyroxine-binding curves for four Species are shown in Figure 1. Binding curves were established in the same manner for all of the species studied. All curves except those for the birds and guinea pigs, show gradually rising levels of protein—bound thyroxine with increasing concentrations of total thyroxine used until a plateau is reached. The plateau indicates saturation of the capacity of TBG for T4. Sodium barbital buffer (pH 8.6) inhibits thyroxine binding to prealbumins. The very high dilutions of 30—35 employed in these experiments reduce even further the usually feeble binding of thyroxine to most mammalian *Standard Error of the mean. 28 29 .mHmEHcm mo mofioomm omnsp was GmEOB SA omB mo mo>Hso mcflpcflnl 3...: cum: J .25» v BII.H wnsmflm O. n _ 0? mm on 0 mm ON 0. O. m _ _n _ a _ I SlilqllII-IOJJIQIIIJO o o o o o o o o J c d c a a a a G 4 I d C d EVE? 3429. o 5:. 342m“. 0 I auuzm 333... a d 2233 d d ON (961) NIELlOHd 01 a/vnoa '1 . I. . ..... .7. ............... n . _ .20... ....r. .‘ufitty w. an...“ _. L1W1¥.E urn... L . : E .l . a. . H i a! I J ’11 allqll - 1 I11 ‘ 30 albumins. The saturation capacities represented by the plateaus are, therefore, almost entirely those of the thyroxine—binding a—globulins since these are the only other thyroxine—binding proteins of consequence. The binding curves in all species studied plateaued at total thyroxine contents ranging from 5-35 ug/lOO m1 serum. So, for these species, total thyroxine concentrations of 35-42 ug/lOO ml serum were chosen for use in the deter- mination of the TBG T4-binding capacities of the sera of individual animals. Thyroid Parameters of Man and Other Species The mean capacities of TBG for T4 with the corres- ponding mean serum thyroxine levels for women, cattle, Sheep, horse, deer, rats, guinea pigs, turkeys and chickens are presented in Table 1. The mean ratios of serum T4: binding capacity or mean Saturation Index (SI) of women and various species are also given. The capacities of TBG for T4 are generally higher than the serum thyroxine levels in man and all ungulates except the deer. The rodents and the birds, however, have higher serum thyrox- ine levels than the capacities of their TBG (if any is present) to bind thyroxine. The difference is succinctly dramatized by the much higher SI in rats (4.06 1 0.41) compared to those of the ungulates and man (p < 0.01). It is not possible to calculate meaningful Saturation Indexes for guinea pigs and birds studied because these 31 .OHmN Eonm unoHoMMHU haDSSOHMHSmHm Do: oDHm>HH .SmoE mEH m0 Hound pnopqmpmH AH0.0 V dc Hm Hoop .m> Hm owwasmcb mo.o H mn.o ”Hoop pmooxm mwumHsmcs Ham mo Hm ..... mN.O OH.H .Omz O HmHmsmHaoav mmxnse ..... OH.O me.H .mmz OH AmHmmquocv mGOMO Hflv ..... NH.O H em.m H..mmz OH HOHE mmaHso HO.O H OO.O HH.O H ON.O mm.O H OO.H OH mHmm HH.O H OO.N mH.O H OH.OH O0.0 H ON.O OH meO OH.O H OO.O Om.O H mm.m Om.O H OO.m m mmmHom NO.O H OH.O Om.O H OO.NH es.O H NO.mH O mHmou HSOHE e0.0 H HO.O OH.H H HO.NH m0.0 H mO.mH O modem OO.O H OH.O OO.O H OH.O NH.O H mm.O OH mHmHHmm mm.O H NO.O mm.O H ON.NH .ON.H H mO.eH HH cages Honum Hadwwm Afisuow HE 00H\qa may monEmm Ausoamoumcozv .OHm H xmeaH Hs OOH\ a mac HoHHm HHHommmo ome Ho .02 mmHowdm SOHHMHsumm coo: .Upm H we Eduwm and: maflocflmlva :moz .mHmEHcd mo mowowmm msowum> cam Gmfioz mo mnouoamumm UHOHMEBII.H mqmda 32 showed negligibly low levels of thyroxine-binding capacity. Mean capacity of TBG for T4 in horses is 3.60 i 0.36 ug T4/100 m1 serum. TBG capacity for the other ungulates averaged 12.42 1 1.97 pg T4/100 m1 serum. Horse TBG thus has a relatively lower thyroxine-binding capacity than that of the other ungulates (p < 0.05). Serum thyroxine in the horse'has been previously reported to be primarily bound by albumins. However, from the present data, the TBG Saturation Index is 0.66 (Table 1). indicating that there is more than enough TBG present to serve as carrier for the existing thyroxine. In this respect the horse does not differ from the other ungulates. The white-tailed deer (Odocoileus virginianus) is peculiar among ungulates studied in having a much higher mean Saturation Index of 2.02 1 0.11 compared to the mean for other higher animals of 0.76 1 0.03. The capacity of deer TBG to bind T4 is generally comparable to that of other ungulates. But serum thyroxine levels are generally higher than those of other ungulates. This leaves a sub- stantial level of T4 in excess of the TBG T4—binding capacity. The excess may be bound by one or more other T4 carrier proteins. It has been reported, as cited earlier, that 48 per cent of an administered dose of 131 I-T is bound to albumin and 36 per cent to d-TBG in 4 the Muntjak deer. Our results thus reinforce this earlier finding. 33 Effect of Pregnancy on Serum T4 gpd TBG Capacities Serum thyroxine levels of pregnant sheep, cows, rats, and guinea pigs were slightly higher than in non— pregnant animals (Tables 2 and 3). The capacities of TBG for T4 were also slightly higher during pregnancy than in nonpregnant animals. But, unlike previously cited reports, in man, pregnancy in these animals did not cause any significant elevation in either the serum thyroxine levels or the capacity of TBG to bind T Of perhaps greater 4. significance is the fact that the mean TBG Saturation Indexes (where these could be calculated) of the nonpreg- nant animals were no different from those during pregnancy (Table 2). The mean TBG Saturation Index of 0.81 i 0.07 in nonpregnant sheep is statistically no different from the SI of 0.70 i 0.04 in pregnant sheep. This ratio is also essentially unchanged during pregnancy in cows when compared to the ratio in nonpregnant cows. The rats, which have the very high SI of 4.06 i 0.41 Show only a slight alteration to 2.58 1 0.36 during pregnancy, no significant change even here. Effect of Oral Contraceptives on Serum T4 and TBG CapacitIes Serum thyroxine in eight women taking oral contra- ceptive pills averaged 14.31 1 0.55 ug T4/100 ml serum (Table 4). The mean serum thyroxine for eleven control women was 12.29 1 0.52 ug T4/100 m1 serum. Serum T4 levels .mmp\xHHE 03 MH mo mmmuo>m cm mcHODUOHHHH .hmp\MHHE mx on no mmmum>m cm mSHUSUOHms 34 No.0 H H0.0 Hm.0 H 0m.m mm.0 H 00.0 0 Heusmcmmum mSHHMHomq m0.0 H 00.0 00.0 H Hm.m mm.0 H m>.m m «ammo msflumuomq m0.0 H 05.0 mm.0 H mm.m mm.0 H Nv.0 0m mHoMHon Has 0H.0 H 00.0 00.0 H 00.0 00.0 H b0.0 0 mHmMHoz Hcmcmmum mmmp 00m 00.0 H 00.0 mm.0 H m0.m Hm.0 H Hm.m 0H mHoMHon Hsmcmonm mmmp 00H 00.0 H 0n.0 mv.0 H mm.m 00.0 H 05.0 0H mHmMHmn AHOHHSOOV ammo "mzou OO.O H O0.0 OO.O H mm.mH O0.0 H mm.m O Emmem OOHHmHomH 00.0 H 0b.0 00.0 H 0m.0H mm.0 H nw.HH 0 @003m Hamsmoum no.0 H Hm.0 00.0 H m0.mH OH.H H H0.NH 0 AHOHHSOOV Scmo "momsm v xmcaH Assumm Hs OOH\ H Saw Assumw Ha OOmeav mmHmsmm HmquH Ho GOHHMHSHmm MHHommmo wme mSHchml B B Edumm mo .02 mHMHm HMOHOOHOHmmzm .mBOU was @0050 Hcmcmmnm can mcHHMHomq .cmmo SH meHmEmumm HMGHOHNEB mo HOHHm .pHm H mammzla.~ mqmda 35 .OHmN EOHH HcoHomHHp >HHSMOHHHcmHm Hos oSHm>H lllll .062 00.0 H 00.N OH HemcmmHm ..... ..mwz NH.O H hm.m OH AHoHHeooO ammo umHm moSHDO 0m.0 H 00.0 N.0 H n0.m nm.0 H 00.h b Hamsmoum H0.0 H 00.0 mm.0 H mn.H OH.0 H 0m.0 0H AHOHHGOOV ammo "pom xoch AESHom HE 00H\0B may AESme HE 00H moHQEmm oumum can HwEHc< SOHHMHSHom huflommmo mchaHml0B oma \09 may 09 Esumm mo .02 . . .mmHm mmcst can muom Hamcmwnm can come we meHoEmumm HMUHOHMEB AmHOHHm .Upm Hv cmozll.m mqmde 36 .A00.0 A my 000m so smEoz .m> 0OHHcoo mo 00 00.0H 00.0H 00.0H 00.0H 00.0 H 00.0 H HOHHm .Uum H 00.0 00.00 00.00 00.0 00.00 00.50 :60: 0 0 0 00 00 00 mm0mEmm mo .02 50.0 00.50 05.50 00.0 55.00 00.00 00.0 00.50 00.50 00.0 00.00 05.00 00.0 00.50 00.00 05.0 00.00 00.00 00.0 00.00 00.00 00.0 00.00 50.00 05.0 00.0 00.00 00.0 00.50 00.00 00.0 05.00 00.00 00.0 00.00 00.00 05.0 00.00 00.00 00.0 05.00 50.00 00.0 00.00 00.00 00.0 00.00 00.00 00.0 00.00 50.00 00.0 00.00 00.00 00.0 00.00 00.00 00 0B ESHmm mHHOMQmo 00 0B ESHmm hHHUMQMU 0cH0eHma0e wme mcchHmu0e wme m00Hm m>HHmwomuucou so . . 00HHGOU .mw>0umoomanoo 0MHO mc0xma meAB 0cm 20803 00HHC00 SH AESHmm 0E 000\0B 0:0 m0m>oq oGHxOHMEB Eduwm can Ume mo mw0H00mmmU mSHUQHmIoGHxOH%£BII.0 m0m<9 37 were therefore significantly higher (p < 0.02) in women ‘ taking contraceptive pills compared to T levels in controls. 4 Thyroxine-binding capacities of TBG in women taking the pill averaged 24.19 i 0.92 ug T4/100 m1 serum. Binding capacities in control women averaged 17.45 i 1.20 ug T4/ 100 ml serum. The pill seems to be associated with an even greater increase in the capacity of the TBG for thyroxine (p < 0.01). In spite of these observed changes the rela- tionship between serum thyroxine levels and the T4-binding capacity of TBG did not change significantly. The mean Saturation Index of 0.82 i 0.05 in women not taking the pill is statistically no different from that of women on the pill (p > 0.10), as shown in Table 4. Saturation Index in Thyroprotein- Treated Cows Thyroprotein—treated cows had a mean T4-binding TBG capacity of 10.03 i 0.65 ug T4/100 ml serum (Table 5). This was statistically no different from the mean of 9.39 i 0.41 ug T4/100 m1 serum of cows before thyroprotein treat— ment. Mean serum thyroxine levels were, however, signifi- cantly raised (p < 0.01) by thyroprotein treatment over pretreatment levels (Table 5). Saturation Indexes signi- ficantly higher than those in non-treated cows (p < 0.01) were therefore obtained. Since serum thyroxine levels were greatly increased with only slight corresponding increases in the capacity of T -binding TBG, the values of 4 serum T4 and binding capacity became almost identical. In .A00.0 v my “00 pmpmoHHIQHmHOHmoumgH .m> HOHHSOU 38 .A00.0 v my "08 ESHom pmumouplc0®H0Hm0Hmnu .m> 0OHHSOU .A00.0 A my "mmHHHOMmmo 0GHUS0Q woumonpln0wuoumoumsp .m> 00HHQOU 00.0 H 00.0 H 00.0 H 00.0 H 00.0 H 00.0 H HOHHm .pHm 50.0 00.0 00.00 00.0 00.0 00.0 H c002 00.0 00.0 00.00 00.0 00.0 00.0 00.0 50.5 00.0 00.0 00.0 05.5 05.0 00.0 00.0 00.0 50.0 00.0 00.0 00.00 05.0 00.0 00.0 00.00 50.0 00.0 00.0 00.0 00.0 00.0 00.0 00.00 00.00 00.0 05.0 05.0 00.0 00.00 00.00 00.0 00.5 00.0 00.0 00.0 00.0 05.0 00.0 05.00 00.0 00.0 50.0 00.0 00.0 00.0 H0 09 ssHmm 0HHmmmmo Hm 0e ssumm mHHmmmmo manch-0H manaHm-HH Uwumwuwic0wuoumouhaa A0300 pwwmwhuzbv 00HHQOU .mmsouw EHom mo moxmch SOHHMHSHmm 00 .0300 vmumeHGD paw UmHmeBISHoHOHm Iouth Hcmcmwum mo AESHmm 0E 000\0B 010 0B Esumm .meHHOMmmo 020©c0m|0a me||.0 mqm0wo 00Hmcomz paw Hmzuoz .msumm mo AmHOHHm .mHm HO mmxwqu QOflu.m.HDu.Mm 0mm”. Gwmzll.m maHm/NB 43 during the second and third trimesters are similarly higher (p < 0.05) than the maternal levels during the same periods (Appendix B). In contrast, several workers have reported that human fetuses Show lower serum thyroxine and TBG T4-binding capacities than their mothers. The differences in fetal and maternal thyroid parameters between the human and the bovine probably set the stage for the peculiar thyroidal activities that are observed in bovine neonates. There is a rise in TBG mean Saturation Index of 1.50 i 0.20 on the day the calf is born to 1.98 t 0.27 on the day after birth. Whereas the serum T4 declined from 18.28 i 1.67 Hg T4/100 ml serum on the day of birth to 17.11 i 1.13 one day after birth, the T -binding TBG 4 capacity dropped rapidly from 12.42 i 0.94 Ug T4/100 m1 serum to 9.65 i 0.99 ug T4/100 ml serum during the same period. The net result is liberation of T4 from TBG for metabolism during the first critical days of neonatal life. Avian Thyroid Parameters Thyroxine-binding capacities are negligibly low in male chickens as well as in laying and nonlaying chickens and turkeys (Table l and 3; Appendix H; Table 7). Male turkeys, however, have measurable though low T4- binding capacities of 1.34 i 0.24 Mg T4/100 m1 serum and serum T4 values of 1.51 i 0.21 Hg T4/100 m1 serum. .OHwN Scum quHmMMHU 00HSMOHmHS00m Ho: 050m>H 00.0 H 05.0 00.0 00.0 00.0 H 00.0 5 mmxude 00m: IIIII 00.0 00.0 .002 0 mSOMOHSU @002 IIIII om.o mm.0 .mmz w Hwhmq ..... mN.O OH.H .mmz OH Hmstcoz uwmxHSB IIIII 00.0 00.0 .002 00 Momma IIIII 00.0 05.0 0.002 00 Hmmn0soz "c0x00co xmccH AaaHmm Hs OOH AESHmm He OOH\OH may mm0QEmm wumum paw mOHOGQm :00HmH5Hmm \0B 010 0E Esumw 0H0ommmo mSHUSHmI0B oma 00 .oz .mmeHSB paw mcmx00£o mo mumuofidhmm 0MUHOHMEB AHOHHm .pHm H0 cmeII.5 mamfla 45 Saturation Indexes could thus be calculated only for male turkeys and these were 1.75 i 0.46 pg T4/100 m1 serum. Nonlaying turkeys have 1.18 i 0.25 pg T4/100 ml serum and this value is not significantly different from either the male level above or the laying T4 level of 1.39 1 0.30 pg T4/100 ml serum. Serum thyroxine levels are 2.10 1 0.42 pg T4/100 m1 serum for male chickens and 1.75 1 0.14 pg T4/100 ml serum for nonlayers. There is no significant difference between these two. Layers have 1.00 1 0.16 pg T4/100 m1 serum, a value which is significantly lower than that of either the nonlayers (p < 0.05) or the male chickens (p < 0.01). GENERAL DI S CUS S ION Differences in the thyroid parameters of various species may be related to the adaptations that occurred in each species as it adjusted to particular environ— ments during its evolutionary development. More specifi- cally the differences between the primate and ungulate groups on the one hand and the rodents and birds on the other are related to the binding protein which has the greatest capacity for thyroxine in each group. In previously cited reports it was Shown that d—globulins are the major thyroxine—binding proteins in primates and most ungulates. These findings are borne out by the fact that binding capacities of the TBG in all the higher mammals studied except the deer exeeed the serum thyroxine levels (Table 1). Mean SI values below 1.0 were therefore obtained. In rodents and birds, the meagre, if any, thyroxine—binding globulins that occur have correspondingly lower capacities to bind T4 (Table 1: Appendix G and H) because thyroxine is almost entirely bound by the albumins. The data clearly indicate that when the value of the Saturation Index is less than 1.0, d-globulins are 46 47 the major carriers of thyroxine as is the case in humans and most ungulates. If however, the SI is significantly greater than 1.0 as is the case in deer, rodents and birds, then other proteins, presumably the albumins and prealbumins Abecome carriers for the excess of T4. In this regard Saturation Indexes constitute quite sensitive indicators of the role played by TBG in binding the serum thyroxine of various animals. During pregnancy, the thyroid parameters of sheep, cows and rodents are not substantially altered from those in open animals. This is in contrast to reports in humans that Show Significant increases in both serum thyroxine levels and T4-binding capacities of TBG during pregnancy. The lack of any significant change in the TBG T4-binding capacities of pregnant sheep is in accord with reports by Annison and Lewis (1958). The values reported by these authors range from 12-16 pg T4/100 m1 serum for the T4-binding TBG capacities of nonpregnant Sheep. Our experiments yielded a mean value of 15.65 1 0.65 pg T4/100 ml serum. These results suggest an impor— tant species difference between primates on one hand and lower mammals on the other. The mean Saturation Indexes of the pregnant animals are not significantly different from the indexes of nonpregnant ones. Even in rats, which have a much higher SI, this was only slightly altered during pregnancy. These findings emphasize the constancy 48 of the serum T4: TBG relationship in different physiologi- cal conditions. Data on the effects of contraceptive pills on thyroid parameters of women further bear out this constancy, despite individual variations in the items that determine Saturation Indexes (Table 4). The observed alterations confirm previously reported findings of the effects of contraceptive pills. Standeven (1969) has reported a significant increase of the PBI values in women taking the pill for longer than 29 months. Mishell eppgl, (1969) report slight elevations of PBI values in women within the first week of therapy and a definite further progression upward after one month. Many of the PBI values of such women after one month of therapy werein the hyperthyroid range. Williams gp_§l. (1966) reported significant changes in PBI as early as 14-20 days after the beginning of therapy. All these reports bear out the effect of contraceptive pills on thyroidal activity. What could be responsible for such observed alterations in thyroid indexes in women on con- traceptive pills? AS has already been pointed out in the literature review, investigators have found Significant elevations in the thyroxine-binding capacities of the TBG of humans given diethylstilbesterol that were comparable to those which occur during pregnancy. More recently, Gimlette and Piffanelli (1968) reported Significant increases in total 49 serum thyroxine of both pregnant subjects and those taking oral contraceptive pills but no significant difference between the values of the two groups. Williams EE_EA' (1966) suggested that all the changes occurring in women who took the pill reflected the effects of estrogen on the thyroid hormone binding capacity of serum proteins. By increasing the amount of bound thyroid hormones, estrogen would decrease the relative amount of free thy— roid hormone and so cause raised PBI values. This would be the case if serum thyroxine levels remained unchanged. Our results, however, showed a substantial increase in serum thyroxine levels as a result of pill therapy to match the increase in T4-binding TBG capacities. This truly fits the classical picture of thyroidal activities. In the classical way of looking at thyroid hor- mone-binding protein relationships TBG capacities are raised during contraceptive pill therapy as they are dur- ing human pregnancy probably as a result of elevated levels of plasma estrogen occurring during these periods. The resulting increase in unsaturated TBG causes a lower- ing of free thyroxine (reflected as reduced T3-I131 uptake in the Hamolsky* test). The thyroid-pituitary feedback mechanism then stimulates the production of pituitary thyrotropin (TSH) which causes increased activity of the thyroid gland and the release of more thyroxine into the *Hamolsky and Freedberg, 1960. 50 bloodstream giving rise to increased serum thyroxine and PBI levels. Thus, the two factors tending to regulate free thyroxine are again brought into proper relationship, but at individually elevated levels. This classical scheme is fully borne out by the mean Saturation Indexes of the control and in women taking contraceptives. The control SI value of 0.82 i 0.05 is statistically no dif- ferent from the value of 0.69 1 0.06 obtained in women taking the pill. Most of the contraceptive pills currently in use like all those employed in the works cited above contain relatively small quantities of estrogen. One example, Ortho-Novum*, contains 1 mg of the progestational sub- stance norethindrone (l7-d-ethiny1-l7-hydroxy-4 estren-B- one) together with 0.05 pg of the estrogenic compound, mestranol (ethinyl estradiol 3—methyl ether). Is it possi- ble that such relatively low levels of estrogen would be responsible for the significant increases in serum T4 and TBG capacities that have been observed in our laboratory and reported by several other workers? Alexander and Marmorston (1961) have demonstrated that as little as 0.01 pg of ethinyl estradiol or mestranol will raise PBI values in post-menopausal women treated for one to eleven months. They report that such changes occa- sionally occurred as early as 7 days after therapy. If *Ortho Pharmaceutical Corporation, Raritan, New Jersey. 51 there still were any doubts as to the role of estrogen in the observed changes, Hollander §p_31. (1963) demonstrated that medroxy progesterone acetate, a compound with 40-100 times the progestational activity of norethynodrel and without significant estrogenic activity had no effect upon PBI values in male subjects. Only in combination with some estrogen did medroxy progesterone acetate alter PBI values. It therefore becomes apparent that it is the estrogen component, ethinyl estradiol, used by Hollander and coworkers, by Standeven and involved in our experiments which are responsible for the alterations observed. Thyroid parameters of cows treated with thyropro- tein and during lactation constitute examples of conditions under which the T4: TBG relationship is substantially altered. As can be seen from Table 5, therprotein treat- ment substantially increases serum thyroxine levels with no corresponding elevation of T4-binding TBG capacity. The TBG is therefore more saturated as evidenced by the Significantly higher SI value in these than in untreated cows. The present data also indicate that most of the serum thyroxine in cows is bound by thyroxine-binding globulin for two reasons: 1. When the level of serum T4 rises above the binding capacity of TBG, the excess is rapidly metabolized and cleared as previously 52 mentioned. In animals with other binding proteins, T4 in excess of TBG binding capa- city has been shown to be bound by albumins and prealbumins. 2. Even with elevated thyroxine levels during thyroprotein treatment, the Saturation Index is not significantly different from 1.0. In deer, rodents and birds where other proteins play a major role in binding serum thyroxine, the Saturation Index is substantially higher than 1.0 (Table 1; Appendix G and H). The effects of thyroprotein treatment contrast very sharply with the effects of lactation in cows. During heavy lactation, there is a steep decline in serum thyrox- ine associated with a nonsignificant drop in the T4-binding capacity of TBG. The mean SI is consequently lower here than in nonlactating open cows. A new factor may be responsible for this discrepancy. A marked decrease in the uptake of radioiodine by thyroids of lactating rats has been reported by several workers. This decrease seems to be related to the suc- cessful competition of the mammary glands for iodine dur- ing lactation. Thyroid secretion rates have been shown to fall from 2.58 in control rats to 0.99 pg T4/100 gm body weight/day in lactating rats (Lorscheider, 1970). Other reports have shown lower thyroid secretion rates in lactating compared to nonlactating goats. These reports 53 suggest that during lactation, at least in the rat and the sheep, thyroid glands are relatively poor competitors for iodine when pitted against the mammary glands. A low iodine uptake results in the low thyroid secretion rate and low serum thyroxine levels that have been observed in the reports cited above and in our data. In cows, the intensity of competition for iodine between the mammary and thyroid glands is probably respon— sible for the steep decline in serum T4. These cows were producing an average of 30 kg of milk/day at the time of sampling. At such a high intensity of lactation, the drop in serum T4 was steep enough to significantly change the Saturation Index. With lactating pregnant cows, when the cows were producing about 13 kg of milk/day, the competition for iodine drops to the point where serum T4 levels were virtually back to nonlactating open levels. Since the TBG T4-binding capacity was unchanged in the process SI values return to the level characteristic of open nonlactating cows. In the third trimester, when lactation stops, the competition is removed. This boosts serum T4 levels even closer to those of open cows. Since the binding capacity of TBG remains virtually unaffected, the Saturation Index at this time is again within the range characteristic of open nonlactating controls. The factor that makes all the difference during lactation appears to be the competition for iodine. Sheep 54 are not usually selected for their high milk production. It is possible, therefore, that the intensity of lacta- tion of the sheep studied was lower than that of cows. This would explain the relatively smaller decline in the serum T4 of lactating compared to the non-lactating sheep (Table 2). A corresponding decrease in the binding capacity of TBG leaves the mean Saturation Index of lac- tating sheep virtually the same as that of the nonlactat- ing animals. In iodine deficient rats, there is a compensatory shift to the secretion of a higher proportion of T3 (Heninger and Albright, 1966). If this is also the case during the lactation-induced iodine deficiency in cows and sheep, our methods would not detect the T since, as 3 previously mentioned, the affinity of T3 for carrier pro- teins is much lower than that of T4. Saturation Indexes are also useful in the inter- pretation of data on bovine fetuses, newborn calves and deer which are unique among ungulates. As already pointed out in the "Review of Literature," the functionality of bovine fetal thyroids starts as early as 60 days of age (gestation period: 284-286 days). Functionality seems to increase with the progress of gestation. This is reflected in the fact that both serum T4 and the binding capacity of TBG in third trimester fetuses are elevated over second trimester levels (Figure 2). 55 The fetuses live in an optimal amniotic fluid environment. Their basic metabolic demands would, there- fore, be minimal and so very little T4 would be expended. This also helps in the observed gradual build up of fetal T4 levels. AS earlier pointed out, the serum T4 in cows appears to be almost entirely bound to TBG. If the ele- vated levels of T4 were to exceed the binding capacity of the existing TBG, the excess would either be cleared or would shut off any further T production by the thyroids. 4 Two possible factors militate against this: 1. The physiological characteristics of the existing level of TBG may change in such a way as to allow the same quantity of TBG to bind more T4. This possibility has been raised by many workers. 2. There may be some diffusion of small quanti- ties of fetal T4 in excess of the binding capacity of fetal TBG to the maternal circu- lation and a Simultaneous diffusion of some TBG from the mother across the placental membranes to fetal blood. Fell, gp_gl. (1968) have raised the possibility that some varieties of globulin could cross placental barriers. By some such mechanism, the capacity of TBG for T4 increases in almost perfect unison with the increasing 56 levels of fetal T4 through the last two trimesters of pregnancy. This is well borne out by the fact that the fetal SI is 1.05 and 1.13 in the second and third tri- mesters, respectively. In this regard, the TBG may act as a storage vehicle for the attendant high levels of thyroxine during this period in preparation for life immediately after birth. Thyroxine carrier proteins in the white-tailed deer may also have a storage role. Deer are peculiar among the ungulates studied in having twice as much serum thyroxine as there is the capacity of TBG to bind T4. This is well reflected in the deer Saturation Index of 2.04 1 0.11. The deer used in this study were fed a well-balanced diet and kept in captivity. They were not, however, domesticated and still retained their wild instincts. The blood samples for this study were obtained on December 1, 1970 just as the winter started in earnest. With the cold exposure that deer in the wild have to face, they probably need an extra thyroidal store of thyroxine that can be rapidly drawn upon in times of heavy demand. It is therefore postulated that the thyrox- ine in excess of the capacity of TBG for T4 would be loosely bound to other carrier proteins, presumably albumins, and would be the first line of defense against the cold. Previous studies on the Muntjak deer have revealed that more T4 is bound to albumin than to TBG. Should the albumin-bound thyroxine become depleted, there 57 probably would be a release of the T4 bound to TBG as a second line of defense against cold exposure. For bovine neonates, the period of heavy T4 demand is just after birth. Neonatal thyroxine levels are at least double the maternal levels and exponentially decline during the first 6 days after birth. This contrasts with the reported increase in both TSH and thyroid secretion rate of human neonates. Bovine neonates are morphologi- cally and even physiologically more developed than human neonates. In consequence, calves are probably better equipped to handle the stresses of extrauterine life than are neonates of many other Species. How, Specifically, does the neonatal calf withstand its new environment? Perhaps the most revealing thyroid parameter in this respect is the Saturation Index both £2 23239 and soon after birth. AS indicated in Figure 2 and Table 6, the mean Saturation Index of 1.13 1 0.21 of the third trimester fetus increases to 1.50 1 0.20 on the day of birth and peaks at 1.98 1 0.27 one day after birth. The SI value then exponentially falls to 1.24 1 0.24 on the 6th day after birth. Apparently, there is a rapid meta- bolism of binding protein soon after birth. This accounts for the rise in SI on the day of birth compared to the level in the third trimester ip_pp§£g. Metabolism of TBG sets free the T4 that was bound. Even more TBG is meta- bolized one day after birth with the release of yet more T40 58 The bovine newborn, like that of other mammals, experiences cold exposure. Unlike any other mammals described to date, however, the bovine is able to draw from its extrathyroidal store of thyroxine to meet the demands of this critical period. The large quantities of T4 in excess of the exponentially decreasing capacity of TBG are used for metabolism and thermogenesis soon after birth. In this way, the new born calf is able to with- stand near-freezing temperatures. The concept of an increase in heat production by the bovine neonate has been well documented by Roy gt_§l. (1957). These workers showed a curvilinear increase in the heat production of newborn calves from the first through the 5th day, and then an exponential decline from the 5th through the 8th day after birth (Figure 2). Bovine neonates effectively use their enhanced thermogenesis to raise and maintain their body tempera- ture. So, like the deer in winter, neonatal calves with— stand the extrauterine cold environment by drawing from their extrathyroidal store of T4 as a quick and very early defense. In birds, any changes in thyroidal parameters dur- ing laying would be expected to differ from those during pregnancy in mammals because of two possible reasons: 1. The hormonal and oviductal changes that accompany pregnancy are not exactly dupli- cated during laying. 59 2. In most mammals studied TBG is the major T4 carrier protein. In birds, however, all previous reports and the present data for female birds indicate a negligible presence or total absence of TBG. Most of the thyroxine appears to be transported by albumin. On this account, if the capacity of carrier pro- teins for T4 were influenced by hormonal changes during laying, this would be more profoundly expressed in carrier proteins other than TBG. The finding in male turkeys is a bit startling. A pooled sample of serum from 7 male tur- keys was therefore used to repeat the documentation of a TBG T4-binding curve and this too yielded binding capa- city values that were statistically higher than zero. To the knowledge of this writer, there has been no previous report on the binding capacities of TBG in male turkeys. In the one instance of male turkeys where Satura- tion Indexes could be calculated, these averaged 1.75 1 0.46 and again reflect the same picture as in rodents where albumins are considered the major carriers of T4. The serum thyroxine data of chickens point up the possible effects of estrogen that have been reported by Common E51213 (1948). They report that estrogen tends to suppress thyroidal activity in chickens. From the data, in Table 7, male chickens with presumably minimal quantities of estrogen, show significantly higher serum 60 T4 levels than either the nonlayers or layers. In the same way, nonlayers have higher serum T4 levels than layers, in which estrogen levels are expected to be highest. The generally low serum thyroxine levels in birds are apparently related to the absence or negligible presence of TBG. Albumins bind T4 very loosely and readily release it for metabolism. These protein: T4 relationships pro- bably account for the fact that if T4 is administered to chickens, only a transient effect on metabolic rate is observed (Mellen, 1958; Singh et al., 1968). APPRAISAL OF NEW TBG-SATURATION TECHNIQUE The underlying principle of the new method is that the sum total of endogenous T4, exogeneously added cold T4 131 and the I-labeled T completely saturate the binding 4 sites on the TBG. Excess T4 is bound to resin-impregnated sponges and binding capacities are expressed as ug T4/100 ml serum bound to TBG. In most of the work previously reported, a dose of labeled thyroxine was added to serum, the proteins were separated by electrophoresis and the results were expressed as a percentage of the label that migrated with each of the thyroxine-binding proteins. So far as this writer is aware, none of the previous studies except that of Keane et_§l. (1969) have included the endogenous T4 in their computations. In the other methods, the total thyroxine available for binding was not determined. The thyroxine-binding capacity of TBG could not, therefore, be calculated. In the new technique, three conditions insure that the protein-bound thyroxine is associated almost exclu- sively with TBG: 61 62 1. At pH 8.6 using barbital buffer, thyroxine binding by prealbumins is competitively inhibited by the buffer. 2. At the high dilution factor of 30-35 the usually feeble thyroxine binding by albumin is reduced to a negligible minimum. 3. Equilibration of the reaction mixture at 37°C minimizes thyroxine-binding to all proteins other than TBG. The above principle and conditions have been incor- porated with a system of small volume measurements and equilibration which enhances the accuracy and reliability of the new technique. Evidence of this reliability is the results of 20 determination of the T4-binding capacity of TBG in the serum samples of one animal. A mean value of 12.02 1 0.13 mg T4/1OO m1 serum was obtained. This indi- cates quite a high degree of repeatability. Our method of expressing T4-binding TBG capacity as "ug T4/100 ml serum" has been used by only a few others. Besides, some of those who have expressed results in this way have usually measured PBI. One group of workers who measured the binding capacity of the TBG in nonpregnant sheep reported values of between 12-16 pg T4/100 ml serum. Our study yielded substantially similar mean TBG T4-binding capacity for nonpregnant sheep (15.65 1 0.67 ug T4/lOO ml serum). Even in studies where proportions of T4 bound to each carrier protein were expressed as per cent of the 63 total radioactivity, our results were again in general agreement. Tanabe and his associates (1969) found that among the ungulates the Muntjak deer TBG has a relatively low T4-binding capacity because most of the serum thyroxine was bound to the albumins. Results in our studies rein- force the concept of a proportionately minor T4-binding role in white-tailed deer TBG compared to other ungulates. The mean Saturation Index of 0.73 1 0.03 for ungulates was significantly lower than the deer mean of 2.02 1 0.11. In chickens, where very low, if any, TBG has been reported, our findings confirm the earlier reports. Apparently most of the thyroxine in birds is bound by albumin not globulin. In guinea pigs and rats where reports have indicated that the albumins or prealbumins are the major carriers of thyroxine in serum, our results again show negligible or very low thyroxine-binding capa- cities in TBG. So a comparison of the results of our studies with reports on the same animal groups by other workers show a high degree of qualitative and even quanti- tative agreement. However, the new TBG saturation techni- que is very suitable for routine use and eliminates the drawbacks that have been observed in electrophoretic techniques. It is, therefore, considered an improvement on existing techniques and could become an invaluable investigative tool. 64 Perhaps the most significant single application of data from the new technique is their utility in determin- ing the TBG Saturation Indexes of different animal groups in various physiological conditions. Saturation Indexes reflect the relationship between serum thyroxine levels and the T4-binding capacities of TBG. These indexes seem to be tenaciously maintained at some species-specific constant through various physiological states. In the sheep, for instance, the SI is 0.84 1 0.08 in nonpregnant controls, 0.70 i 0.04 during pregnancy and 0.69 1 0.04 during lactation. Even though the serum thyroxine and T4-binding TBG capacities are individually altered during pregnancy and lactation, their relationship (SI) is main- tained virtually constant. In somewhat abnormal physiolo- gical conditions such as when thyroprotein is administered to the bovine the mean Saturation Index does change from the level characteristic of the Species. Mean SI values much closer to 1.00 are obtained apparently because with the observed increase in serum T4 the TBG is substantially saturated. Saturation Indexes of TBG have also sensitively indicated the role played by the globulin asia thyroxine carrier when compared with other thyroxine-binding pro- teins. In the deer, rodents and birds where mean Satura— tion Indexes are substantially higher than 1.0, the bulk of the thyroxine is apparently bound by albumin as con- firmed by other reports. In humans and the ungulates 65 other than deer, where the Saturation Index is less than 1.0, the capacity of TBG for thyroxine is not normally saturated by endogenous thyroxine and most of it is bound by the globulins. This is again in agreement with several previous reports. The new technique for measuring the T4—binding capacities yields not only the proportion of thyroxine bound by TBG but goes further to quantify it. Results from the new technique are more quantitatively consistent than those from electrophoretic techniques. On the other hand, the great variation in results from electrophoretic methods has been well illustrated in a review by Woeber and Ingbar (1968). The reliability of the new technique is in some measure due to the clean and quantitative separation of bound from unbound thyroxine by the resin-impregnated sponges. Another factor that aids in the accuracy of this technique is the virtually complete blocking of thyroxine- binding by albumin and prealbumin. Clearly then, the new technique for measuring T4-binding TBG capacities not only eliminates the drawbacks of most current techniques but also has some new advantages. SUMMARY 1. A new technique for measuring the Saturation capacity of TBG to bind endogenous, radioactive and exo- genously added cold thyroxine (T4) has been presented. The conditions of this method block T4-binding to preal- bumins and albumins and resin-impregnated sponges are employed to separate protein-bound from unbound T4. The- new technique is suitable for routine use and avoids the major drawbacks of electrophoresis. Results obtained indicate good repeatability and are in good qualitative and quantitative agreement with data from previous reports by workers who used other methods. 2. Thyroxine-binding TBG capacities are generally higher than serum T4 levels in man and most ungulates. ’In rodents and birds, the capacities are lower than serum T4 levels because, as has previously been reported, in these groups most of the thyroxine is bound by albumins or prealbumins. This is well reflected in the higher ratio of serum T4: TBG capacity (Saturation Index) in rodents and birds compared to those of humans and the ungulates. 66 67 3. Thyroxine-binding globulins appear to be the only T4-carrier proteins in the bovine. Thus when there is an increase in serum thyroxine in excess of the bind- ing capacity of TBG, the excess is rapidly metabolized and cleared as observed in thyroprotein-treated cows. 4. Unlike the case in man, pregnancy in cows, sheep, and rats is not accompanied by a significant increase in serum thyroxine-binding capacity of TBG. The slight alterations in serum T4 which occur during pregnancy are accompanied by equally slight alterations in the capa- city of TBG to bind thyroxine. Mean Saturation Indexes during pregnancy therefore remain at the levels character-‘ istic to each species. 5. During lactation in cows and sheep, the pre- viously reported decrease in serum thyroxine is accompanied by a nonsignificant decline in the capacity of TBG for T4. In sheep, both parameters are equally depressed. The mean Saturation Index of lactating cows is consequently lower than that of the nonlactating controls, whereas this index is unchanged in sheep. 6. Women on oral contraceptive pills show signi- ficant increases in both their serum thyroxine and capa- cities of TBG to bind T4 when compared to those not taking the pills. Both groups of women thus yield substan- tially similar mean Saturation Indexes. 68 7. Serum thyroxine and TBG T4—binding capacities of bovine fetuses gradually rise at least from the second trimester up to term. Since the T4 levels and binding capacities of TBG are nearly identical during this period there is little or no unbound thyroxine and the Saturation Index of second and third trimester fetuses are 1.05 1 0.05 and 1.13 1 0.21, respectively. At term, the neonatal thyroxine level is 3—4 times the adult level. With the rapid decline of binding capacities of TBG after birth, the thyroxine freshly released from binding is available for producing heat which is necessary at this time to counteract the extrauterine cold exposure of the first few days of life. 8. Deer are unique among the ungulates studied in having very high serum T4 levels and Saturation Indexes. The high indexes indicate that substantial amounts of T4 are carried by a protein or proteins other than TBG. 9. Laying chickens have lower serum thyroxine levels than nonlayers whose T4 levels are lower than those of male chickens. 10. Male turkeys are unique among the birds studied in having TBG T4-binding capacities that are significantly higher than zero. APPENDIX A THYROID PARAMETERS OF DEER, PIGMY GOATS AND HORSES Deer T4-binding Serum T . 4 TBG CapaCity Sample (ug T4/100 m1 (ug T4/100 m1 Saturation Code No. Serum) Serum) Index 589 16.72 10.68 1.57 620 14.28 10.93 1.31 604 16.37 10.39 1.58 602 14.22 6.01 2.37 600 17.97 11.88 1.51 617 14.78 9.05 1.63 603 18.01 11.63 1.55 623 13.54 5.63 2.40 619 15.65 8.40 1.80 614 11.37 6.63 1.71 11 15.52 6.65 2.33 12 17.17 7.13 2.41 616 16.13 6.19 2.61 572 15.83 9.22 1.72 580 16.03 7.29 2.20 574 17.81 7.20 2.47 50611 17.72 8.57 2.07 582 19.84 6.15 3.23 589 18.22 7.80 2.34 Means 1 16.17 1 0.45 1 0.46 2.04 1 Std. Error Sample Code No. GC-A GC-A2 GC-A3 GC-30 GC-32 GC-34 GC-36 GC-38 GC-43 1 Means 1 Std. Error Sample Code No. 205595-6 205581 205501 205588 205550 Means 1 Std. Error 70 Pigmy Goats T4-binding Serum T . 4 TBG CapaCity (Hg T4/100 m1 (pg T4/100 m1 Saturation Serum) Serum) Index 12.91 14.81 0.87 9.03 11.22 0.80 10.47 14.71 0.71 13.51 18.27 0.74 11.38 16.40 0.69 13.48 18.50 0.73 13.23 16.95 0.78 13.87 17.62 0.79 10.85 14.77 0.73 12.08 1 0.57 15.92 1 0.77 0.76 1 0.02 Horses T4—binding Serum T . 4 TBG CapaCity (pg T4/100 m1 (pg T4/100 m1 Saturation Serum) Serum) Index 1.45 4.51 0.33 3.19 2.47 1.29 1.00 4.05 0.40 2.15 3.88 0.48 2.50 3.10 0.78 2.22 1 0.30 3.60 1 0.36 0.66 1 0.18 APPENDIX B THYROID PARAMETERS OF COWS IN VARIOUS PHYSIOLOGICAL CONDITIONS Control Heifers (Average: 17 months old) T4-Binding Serum T4 TBG Capacity Sample (Ug T4/100 m1 (ug T4/100 m1 Saturation Code No. Serum) Serum) Index 431 6.38 7.32 0.87 434 5.61 9.21 0.60 1035 9.72 10.10 0.96 1036 5.71 9.38 0.61 1038 7.05 11.80 0.60 429 7.30 9.84 0.74 1039 6.41 12.03 0.53 1041 4.95 9.33 0.53 1045 6.16 9.91 0.62 1046 8.58 9.41 0.91 Means 1 0.46 1 0.42 0.70 1 Std. Error Lactating Open Cows (Average age 6 years) Std. Error T4-Binding Serum T . 4 TBG CapaCity Sample (ug T4/10O m1 (ug T4/100 m1 Saturation Code No. Serum) Serum) Index 800-6 3.14 6.87 0.45 319—C 3.08 7.07 0.43 796-C 3.52 9.95 0.35 904-C 5.22 8.84 0.59 716-C 3.81 9.84 0.38 Means 1 0.39 1 0.66 0.44 1 Sample Code No. 952-A 902-A 869-A 838-A 811-A 702-A Means 1 Std. Error (Average 180 days pregnant, age 18-24 months) Sample Code No. 561 554 556 555 559 558 546 567 551 557 Means 1 Std. Error (79 days pregnant, average age 5 years) 72 Lactating Pregnant Cows Serum T (ug T4/1OO m1 Serum) 4 (ug T4/100 m1 Serum) .65 5.71 5.70 6.14 4.39 5.52 6.46 1 0.29 T4-Binding TBG-Capacity (pg T4/100 m1 Serum) Saturation Index 8.92 9.09 9.73 7.95 9.47 10.05 9.20 1 0.31 Dry Pregnant Heifers Serum T4 5. 91 5.78 4.94 6.92 4.92 6.07 6.58 6.40 7.17 6.22 4.14 1 0.31 T4—Binding TBG Capacity (ug T4/100 m1 Serum) 0.64 0.63 0.63 0.55 0.58 0.64 .61 1 0.02 Saturation Index 8.73 9.65 6.75 11.04 9.18 8.63 7.79 8.46 9.99 10.11 9.03 1 0.39 0.66 0.51 1.03 0.45 0.66 0.76 0.82 0.85 0.62 0.41 0.68 1 0.06 73 Dry Pregnant Heifers (264 days pregnant on average, age:l8-22 months) T4-Binding Serum T4 TBG Capacity Sample (ug T4/100 m1 (Ug T4/100 ml Saturation Code No. Serum) Serum) Index 549 7.24 11.38 0.64 562 5.14 8.78 0.58 550 7.89 8.18 0.96 563 8.10 8.04 1.01 560 7.00 8.99 0.78 564 4.66 8.96 0.52 Means 1 6.67 1 0.59 1 0.49 0.84 1 Std. Error APPENDIX C BOVINE FETAL THYROID PARAMETERS DURING THE SECOND AND THIRD TRIMESTERS OF PREGNANCY 180-Day-Old-Fetus T4-Binding serum T4 TBG Capacity Sample (pg T4/100 m1 (pg T4/100 m1 Saturation Code No. Serum) Serum) Index 561 6.62 5.65 1.17 554 13.32 11.04 1.21 556 8.89 8.42 1.06 555 13.26 12.11 1.09 559 12.81 11.48 1.12 558 10.26 11.99 0.86 546 14.66 15.08 0.97 567 14.58 11.40 1.28 551 10.23 12.67 0.81 557 13.56 14.06 0.96 Means 1 11.82 1 0.85 11.39 1 0.86 1.05 1 0.05 Std. Error 74 75 264—Day-Old Fetus T4-Binding Serum T4 TBG Capacity Sample (Hg T4/100 m1 (ug T4/100 m1 Saturation Code No. Serum) Serum) Index 549 18.68 13.70 1.36 566 8.72 17.89 0.49 562 20.18 9.05 2.23 550 17.39 14.44 1.20 563 15.76 15.87 0.99 560 16.23 19.85 0.82 564 15.57 18.97 0.82 Means 1 16.08 1 1.37 15.68 1 1.40 1.13 1 0.21 Std. Error THYROID PARAMETERS OF SHEEP DURING VARIOUS Sample Code No. 8-38 8-42 8-04 8-21 8-26 8-25 Means 1 Std. Error Sample Code No. APPENDIX D PHYSIOLOGICAL CONDITIONS Open Nonlactating Sheep Serum T4 (pg T4/100 m1 Serum) 4-35 7-29 4-13 4-33 7-23 4-29 Means 1 Std. Error 16.34 13.15 13.97 11.16 12.99 8.05 12.61 1 1. 14 T4-Binding TBG Capacity (pg T4/100 m1 Serum) 15.56 14.71 14.65 16.69 18.32 14.00 15.65 1 0.65 Pregnant Sheep Serum T4 (Mg T4/100 m1 Serum) 10.53 10.91 8.19 12.47 13.94 12.79 11.47 1 0. 83 76 T4—Binding TBG Capacity (Hg T4/100 m1 Serum) Saturation Index 14.67 15.24 15.32 16.15 17.37 18.81 16.26 1 0.64 1.05 0.89 0.95 0.67 0.71 0.57 0.81 1 Saturation Index 0.72 0.72 0.53 0.77 0.80 0.68 0.70 1 77 Lactating Sheep Std. Error T4-Binding Serum T . 4 TBG CapaCity Sample (pg T4/100 ml (pg T4/100 m1 Saturation Code No. Serum) Serum) Index 4-35 7.71 12.37 0.62 7-29 8.75 13.11 0.67 4-13 10.49 14.79 0.71 7-23 10.83 13.03 0.83 3-06 10.83 14.90 0.73 6-30 7.30 12.95 0.56 Means 1 0.66 13.53 1 0.43 0.68 1 0.04 APPENDIX E THYROID PARAMETERS OF NEWBORN CALVES FROM THE lsT TO THE 6TH DAY OF BIRTH Thyroid Parameters on Day of Birth T -Binding Serum T 4 . 4 TBG CapaCity Sample (Hg T4/100 m1 (Hg T4/100 m1 Saturation Code No. Serum) Serum) Index 405 10.36 10.80 0.96 431 14.00 9.10 1.10 1031 24.71 11.60 2.13 408 22.77 12.43 1.83 994 27.87 9.43 2.95 420 15.71 10.82 1.45 427 17.61 18.34 0.96 417 16.73 13.76 1.22 406 17.18 16.48 1.04 413 15.82 11.44 1.38 Means Std. Error 18.28 1 1.67 12.42 1 0.94 1.50 1 78 79 Day-old Calves Std. Error T4-Binding Serum T . 4 TBG Capac1ty Sample (pg T4/100 m1 (pg T4/100 m1 Saturation Code No. Serum) Serum) Index 405 11.56 10.71 1.08 431 21.04 7.96 2.64 1031 19.12 9.62 1.99 408 18.14 5.44 3.33 994 22.51 6.91 3.25 420 16.16 10.31 1.57 427 19.38 13.09 1.48 417 16.09 14.85 1.08 406 14.54 11.83 1.23 413 12.51 5.81 2.15 Means 1 17.11 1 1.13 9.65 1 0.99 1.98 1 0.27 Std. Error 2-day-old Calves T4-Binding Serum T . 4 TBG CapaCity Sample (pg T4/100 m1 (pg T4/100 m1 Saturation Code No. Serum) Serum) Index 405 12.80 10.22 1.25 431 14.66 5.94 2.47 1031 17.55 9.03 1.94 994 11.33 4.37 2.59 420 14.15 10.79 1.31 427 12.11 9.61 1.26 417 15.86 12.41 1.28 406 14.55 11.61 1.25 Means 13.68 1 0.77 1 0.91 1.65 1 Sample Code No. 3-day-old Calves Serum T (Hg T4/100 m1 Serum) T4-Binding TBG Capacity (pg T4/100m1 Serum) 405 431 1031 408 994 420 427 417 406 413 Means Std. Error Sample Code No. 4. 405 431 1031 994 420 427 417 406 413 Means Std. Error + 11.4 9.92 11.35 14.54 12.55 10.58 9.55 10.26 13.57 11.85 10.07 2 1 0.53 9.21 6.80 9.52 5.83 4.92 10.02 8.31 12.39 9.41 5.71 1 0.74 4—day-old Calves Serum T (pg T4/100 m1 Serum) T4-Binding TBG Capacity (pg T4/100 m1 Serum) Saturation Index 9.48 10.16 9.74 11.29 9.18 7.70 8.42 10.91 10.58 5.22 1 0.61 8.36 4.74 8.64 3.94 8.55 9.25 10.51 10.95 4.39 1 0.83 1.08 1.67 1.53 2.15 2.15 0.95 1.23 1.10 1.26 1.76 1.49 1 0.13 Saturation Index 1.22 2.05 1.31 2.33 0.98 0.91 1.04 0.97 1.19 1.41 1 0.17 81 5-day-old Calves T4-Binding Serum T4 . TBG Capacity Sample (pg T4/100 m1 (pg T4/100 ml Saturation Code No. Serum) Serum) Index 405 10.40 9.16 1.14 431 7.90 4.96 1.59 1031 12.35 8.50 1.45 408 8.15 5.47 1.49 994 6.73 3.56 1.89 420 6.37 8.61 0.74 427 6.66 8.09 0.82 417 11.08 12.95 0.86 406 6.82 5.55 1.23 413 3.52 4.69 0.75 Means 1 7.80 1 0.83 7.15 1 0.89 1.20 1 0.13 Std. Error 6-day-old Calves T4-Binding Serum T4 TBG Capacity Sample (pg T4/100 m1 (pg T4/100 m1 Saturation Code No. Serum) Serum) Index 405 7.40 6.15 1.20 431 7.47 4.30 1.74 1031 13.60 8.61 1.58 408 7.12 4.37 1.63 994 6.99 4.22 1.66 420 8.60 7.87 1.09 427 8.11 9.65 0.84 417 10.76 10.60 1.02 406 4.40 5.67 0.78 413 2.90 3.55 0.82 Means 1 7.74 1 0.94 6.50 1 0.80 1.24 1 0.12 Std. Error . APPENDIX F REPEATABILITY STUDIES: TBG T -BINDING 4 CAPACITIES AND SATURATION INDEXES OF SERUM SAMPLES FROM ONE HEIFER Serum Thyroxine T4—Binding TBG Capacity Saturation (pg T4/100 m1 Serum) (pg T4/100 m1 Serum) Index 6.41 11.92 0.54 6.41 11.92 0.54 6.41 11.90 0.54 6.41 11.95 0.54 6.41 11.39 0.56 6.41 12.66 0.50 6.41 12.67 0.51 6.41 12.60 0.51 6.41 11.62 0.55 6.41 11.95 0.54 6.41 11.19 0.57 6.41 11.49 0.56 6.41 12.47 0.51 6.41 12.54 0.51 6.41 11.11 0.58 6.41 11.48 0.56 6.41 12.96 0.49 6.41 12.83 0.50 6.41 11.53 0.56 6.41 12.14 0.52 Mean 1 Std. Error 12.02 1 0.13 0.53 1 0.01 82 APPENDIX G THYROID PARAMETERS OF RODENTS Nonpregnant Control Rats T —Binding Serum T 4 . 4 TBG CapaCity Sample (pg T4/100 ml (pg T4/100 ml Saturation Code No. Serum) Serum) Index Rpl 6.66 1.51 4.41 Rp2 6.31 1.23 5.13 Rp3 6.62 1.65 4.01 Rp4 6.40 1.37 4.67 Rp5 5.62 1.09 5.16 Rp6 6.15 2.48 2.48 Rp7 5.71 1.05 5.44 Rp8 5.99 1.23 4.87 Rp9 5.46 2.47 2.21 R-l 7.13 3.26 2.18 Means 1 6.20 1 0.17 1.73 1 0.23 4.06 i 0.41 Std. Error 83 84 Pregnant Rats T4—Binding Serum T4 TBG Capacity Sample (pg T4/100 m1 (pg T4/100 ml Saturation Code No. Serum) Serum) Index RC1 5.81 1.74 3.39 R02 7.80 2.45 3.18 RCS 7.80 2.48 3.15 RC6 7.29 2.36 3.09 RC7 6.69 1.22 5.48 RC8 6.57 1.59 4.13 Rp12 7.04 2.66 2.65 Means 1 7.00 1 0.27 2.07 1 0.21 3.58 1 0.36 Std. Error Guinea Pigs Nonpregnant Controls Pregnant Guineajpigs Serum Thyroxine Serum Thyroxine Sample (pg T4/100 m1 Sample (pg T4/100 m1 Code No. Serugg Code No. Serum) A-169 1.83 A-29 0.49 A—l68 2.22 A-167 2.51 A-77 2.89 A—166 2.68 A—105 2.39 A-l65 2.94 A-102 2.63 A—163 1.78 A—78 2.40 A-150 2.68 A-97 0.89 A-145 2.55 A-99 4.21 A-144 2.12 A-108 3.63 A-143 2.34 Mean 1 2.37 1 0.12 2.44 1 0.44 Std. Error APPENDIX H AVIAN THYROID PARAMETERS Male Turkey T4—Binding Serum T4 TBG Capacity Sample (pg T4/100 m1 (pg T4/100 m1 Saturation Code No. Serum) Serum) Index TMl 1.52 1.65 0.92 TM2 1.06 1.92 0.55 TM3 1.09 2.17 0.50 TM4 0.81 0.58 1.39 TM5 1.62 1.55 2.79 #2 2.31 0.64 3.61 #3 2.14 0.86 2.49 Mean + 1.51 1 0.21 1.34 1 0.24 1.75 1 0.46 Std. Error 85 86 Female Turkey (Negligible T —Binding TBG Capacities) 4 Nonlayers Layers Serum Thyroxine Serum Thyroxine Sample (“9 T4/100 ml Sample (pg T4/100 ml Code No. Serum) Code No. Serum) TNLl 0.74 TLl 0.52 TNL2 1.16 TL2 1.16 TNL3 0.16 TL4 0.59 TNL4 1.13 TL5 0.94 TNL5 1.55 TL6 0.74 TNL6 1.12 #6 2.68 TNL7 0.99 #7 2.17 TNL8 2.60 #8 2.34 Mean 1.18 1 0.25 1.39 1 0.30 Std. Error F . I u;\..q‘.. 87 00.0 a 00.H mm.a 0130 04.0 016 an.a 01o 00.0 010 00.0 ego mm.0 mqo 00.0 ego 00.1 mqo 00.0 who 00.1 110 Afidnmm HE .oz OUOU 00H\0e may mamsnm «B Enumm mehmq Aao.o v my mnmmmacoz .m> mumhmq Amo.o v my mummchoz .m> moan: Hopum .pum VH.0 H mh.H Nv.o H OH.N H COTE 00.1 0quo 00.N maze mm.a 0120 00.0 Haze 00.H 0azo 00.1 0Hzo Hm.H GHZO mh.o mEU 00.0 quo 00.0 02o 00.0 0126 00.0 020 Hw.H MHZU mv.m MED 00.H quo 00.0 «So mh.N HHZU MN..N H20 AEDHmm HS .02 OCOU AESHmm HE .OZ OUOU n a 001\ee may he gem 001\ee 01V he saw as Eupwm we Eupom muommagoz mmawz v Amwflpflommmo wme mcflpcflml mCOMOHso e panamaamwzv APPENDIX I SATURATION TECHNIQUE FOR MEASURING BINDING CAPACITY OF TBG N/10 HCl Solution Weigh out 9.856 gm of concentrated HCl solution (37% HC1)- Dilute to 1 L using distilled water. Standardize with Na CO 2 3 solution using phenolphthalein indicator. Adjust to 0.1N if not at the correct pH. Label this solution B. M/10 Sodium Barbital Solution Weigh out 20.618 gm of powdered sodium barbital. Dissolve in distilled water and dilute to 1 L. Label this solution A. Barbital Buffer pH 8.6 Add 12.1 ml of solution B from a burette into a 1000 m1 volumetric flask. Dilute to 1000 ml with solution A. Mix thoroughly by inverting the stoppered flask several times. Check the pH of the buffer solution using a pH meter. The pH should be 8.6. 88 89 Stock Cold T4 Solution Weigh out 10 mg of pure L-T4 and transfer to a 100 m1 siliconized volumetric flask. Dissolve in glass distilled water with the aid of a minimum quantity of NaOH solution and dilute to 100 ml to give a 100 pg T4/ml solution. Pipette out 1 m1 of this solution to dilute to 20 ml with glass distilled water to give a stock 5 pg T4/m1 standard. Various working standards can be prepared by aqueous dilution of the stock solution. Working standard concentrations varied from 0.1—0.5 pg T4/ml depend— ing on the endogenous T4 level of serum being investigated. 131I-L-T4 Solution Portions of the stock radiothyroxine solution are diluted such that a volume of 0.05 ml of the working standard gives 2,000-40,000 Cpm. Polypropylene Tubes and Resin-impregnated Sponges Each polypropylene tube has the following dimensions: 1.3 cm I.D. X 8.6 cm (Abbott Radiopharmaceuticals, North Chicago, Illinois). Polyurethane resin-impregnated sponges* capable of adsorbing free T4 have the following dimensions: *In the absence of resin—impregnated sponges the technique can be modified to allow the use of IRA—400 anion—exchange resin. 90 1.1 cm O.D. X 1.95 cm (Abbott Radiopharmaceuticals, North Chicago, Illinois). 7. Procedural Sequence a. Measure into each of 3 polypropylene tubes the volume of 0.087M sodium barbital buffer (pH = 8.6) which sums up with the combined volumes of 1311- labeled and unlabeled T4 solutions as well as serum to give a total volume of 2.0 - 2.5 ml and a serum dilution of 30-35. Two of these tubes are dupli- cates, the third will be a serum—free blank. The amount of endogenous T4 in the serum volume used in duplicates is exactly replaced by cold T4 in the blank. b. In sequence, measure the calculated volumes of labeled T4, cold T4 and lastly serum (see Appendix I-9, equations 1-4) into the buffer using a syringe microburet.* After the addition of each of the three sources of T4, the external tip of the syringe needle used is rinsed with 2—4 drops of barbital buffer in order to effect complete quantitative transfer of whatever is measured. 0. To ensure complete equilibration of all ions in each polypropylene tube, the liquids are vortex- mixed for 15 seconds after the addition of either cold or radioactive T4 solution, and 30 seconds after the addition of serum. *Micro-metric Instrument Corporation, Cleveland, Ohio. 91 A11 tubes are then incubated for 60 minutes in a 37°C water bath. The tubes are taken out of the bath and a resin- impregnated sponge is immediately added to each tube. Each sponge is gently depressed three times with a plastic plunger. Initial radioactivity (CPM) from each tube is determined using a scintillation well counter and gamma ray spectrometer set for counting at the 1311 peak. The tubes are again returned to the 37°C water bath for 30 minutes and then taken out. Each tube is immediately filled with distilled water. The sponges are each washed 3 times with distilled water using a suction apparatus and Special plastic aspirators for suCking out the liquids. A final count of radioactivity from each tube is then taken. Special Critical Steps in Procedure» 1. A11 syringes and needles used in this technique have to be specially cleaned using in sequence the following: Aqueous Alconox Solution, Radiac wash (for radioactive syringes and needles). Tap water. 92 Glass distilled water. Ethyl alcohol (for dissolving and washing off any thyroid hormone). The needle and syringe are washed six times using each of the above liquids. 2 A11 instruments so washed are dried in an oven and cooled to room temperature before use. 2. When adding thyroxine solutions or serum to special polypropylene tubes (Tetrasorb-125 Kit) care must be taken to let the drops of solution fall to the bottom of the tube. The drops should not be allowed to run down the sides of the tubes. If some drops do run down the tube wall, their path must be rinsed down with 2-3 drops of barbital buffer. This ensures that there is no significant adsorption of thyroxine to the sides of the polypropylene tubes. 3. A11 thyroxine solutions and serum must be added into a buffer medium. A reversal of the order detailed above results in binding of therxine to other proteins in addition to TBG. Computation of the Concentrations of Thyroxine to be Employed in Each Experiment The total quantity of thyroxine needed to saturate the thyroxine-binding capacity of TBG in serum of a particular animal species is read from the flat part of the binding curve as set forth in the results sec- tion. This concentration inCludes the endogenous 93 thyroxine of the serum in question which is determined by the Tetrasorb-125 resin-sponge technique. Some 131I-labeled thyroxine is employed as a tracer. Since the combined endogenous and labeled thyroxine in the amounts used are usually insufficient to saturate the serum binding proteins, some cold thyroxine is also added. In the subsequent reactions, the optimal quantity of serum used has been found to be in the order of 0.07 ml. The contribution of endogenous T4 is obtained by the equation a x b _ (l) 100 ‘X where, X = pg T4. a = serum volume in m1, and b = serum T4 concentration in pg%. Amount of 131I-T4 contributed by the labeled T4 solution is given by the equation y = a1 x b1 (2) where, . l y = pg ;3 I-T4. a1 = volume of T4 solution used, m1. bl = concentration of 131I-T4 solution, pg/ml. 10. 94 Sum of T4 from serum and 131I-T4 solution = (x + y) Hg Amount of cold T4 needed is given by m = Z - (x + y) (3) where, m = amount of cold T4, pg. N ll amount of total T4 (pg) needed to saturate binding sites on TBG. Then, volume of cold T4 solution needed is given by the equation a2 = m/b2 (4) where, a2 = volume of cold T4 solution, m1. b2 = concentration of T4 solution, pg/ml. Correction for Thyroxine Not Bound by Resin Sponges Since the resin-impregnated sponges do not have an unlimited capacity to bind free thyroxine, blanks are employed for each level of thyroxine. The blanks have the same amount of thyroxine as corresponding dupli- cates but no serum. The difference between the first and second counts of radioactivity of the blanks gives the amount of thyroxine not taken up by the sponges. This difference is calculated as a percentage of the 95 initial count and the percentage is then used to correct final counts of serum-containing tubes as follows: The figures used in this illustration are part of the results of an actual experiment. Initial Final counts/min cpm. Difference % Difference Blank 13,544 12,213 1,331 cpm 1331 = Iggzleoo 9.83 Serum— 13,669 6,737 containing tube From the blank, the final cpm of the serum-containing tube was underestimated by l2§§§1§02;§§ = 1344 cpm. The corrected final count should, therefore, be 6737 + 1344 = 8081. It is the corrected final count that is used in the calculation of the pg T4 bound to protein. 11 Computation of the Capacipy of TBG for Thyroxine The basic principle of this technique is that thyroxine-binding globulin will bind both labeled and unlabeled thyroxine to saturate all its binding sites at 37°C. The thyroxine in excess of the maximal bind- ing capacity of carrier proteins at the temperature employed is bound to the resin-impregnated sponges. After the sponges are washed, only the non-protein- bound thyroxine is left bound to them. The difference in radioactivity between the initial and corrected 12 96 final counts is therefore attributable to protein bound thyroxine. The initial count represents the total thyroxine used in the experiment. From this, the number of counts that represent one pg of thyroxine can be calculated. A knowledge of the counts per pg T4 enables a calculation of the pg of T4 bound to protein from the difference between the initial and final counts. Final results as pg% T4 bound to TBG are calculated by the equation, x 1&9 (5) ole where, a = pg % T4 bound to TBG. b = radioactivity bound to TBG, cpm. c = initial cpm/pg total T4. d = serum used, m1. Saturation Index of Thyroxine—Binding Globulin The term "saturation index" (S1) was adopted as a concise description of the relationship between serum thyroxine levels and TBG T4—binding capacities. "Saturation" was considered appropriate since the T4- binding capacities were obtained when serum samples were flooded with such concentrations of thyroxine as would more than saturate the binding capacities of the thyroxine-binding globulins. The Saturation Index is calculated according to the equation, 97 (6) Saturation Index = S C where S serum T4 pg/lOO ml. C = T4-binding TBG capacity pg/100 m1. Significance of differences within and between various animal species was obtained by the student "t" test (Li, 1964). The Chauvenet'criterion* was employed to determine whether or not extreme values of a series should be discarded. 13. Rationale of Procedure The pH of sodium barbital buffer as well as the 30-35 times dilution of serum create conditions which virtually block the binding of thyroxine by carrier proteins other than TBG. Incubation at 37°C simulates the average body temperature of most mammals. In order for data from other animal classes to be comparable, their sera are also incubated at 37°C even though the normal body temperature of these animals might differ from 37°C. A maximum of 60 minutes is required for all the available binding sites on the a-globulin to become filled by thyroxine. In several preliminary experiments it was found that 30 minutes is sufficient for free thyroxine binding by the sponges. *Documenta Geigy, Scientific Tables, 5th Edition, Basle (Switzerland): S. Karger, New York, p. 47, 1959. 98 Since the sponges have a maximum affinity for thyroxine of about 0.17-0.20 g, blanks or controls were run for each concentration of total T4. Whatever excess of free T4 is left unbound by the sponges in the blanks, is calculated as a percentage of the total T4 used. The tubes containing sera are each corrected by this percentage excess as explained in the section on calcu- lations. The initial count represents the radioactivity of both the protein bound and unbound thyroxine. After the sponges are washed three times all the protein- bound thyroxine (labeled and unlabeled) is removed in the washings. The final count represents the radio- activity of the thyroxine that was in excess of the saturation capacity of the binding proteins. Neither the binding proteins nor the resin sponges can distin- guish between the labeled and unlabeled thyroxine. 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