Study of Four New Kindreds with Inherited Thyroxine-Binding Globulin Abnormalities POSSIBLE MUTATIONS OF A SINGLE GENE LOCUS

Five families with inherited thyroxine-binding globulin (TBG) abnormalities were studied. On the basis of serum thyroxine (T(4))- binding capacity of TBG in affected males, three family types were identified: TBG deficiency, low TBG, and high TBG capacity. In all families evidence for X-linked inheritance was obtained and in one family all criteria establishing this mode of inheritance were met. Only females were heterozygous, exhibiting values intermediate between affected males and normals. Overlap in heterozygotes was most commonly encountered in families with low TBG.QUANTITATIVE VARIATION IN THE SERUM CONCENTRATION OF FUNCTIONALLY NORMAL TBG WAS DEMONSTRATED BY: (a) failure of serum from TBG-deficient subjects to react with anti-TBG antibodies; (b) normal kinetics of T(4) and triiodothyronine-binding to TBG in sera from subjects with low TBG and high TBG capacity; (c) concordance of estimates of TBG concentration by T(4) saturation and by immunological methods; and (d) normal rate of heat inactivation of TBG.No abnormalities in serum transport of cortisol, testosterone, aldosterone, or thyroxine bound to prealbumin could be detected.These observations suggest that all the TBG abnormalities thus far observed reflect mutations at a single X-linked locus involved in the control of TBG synthesis.     

Dependence of the thyroxin/thyroxin-binding globulin (TBG) ratio and the free thyroxin index on TBG concentrations

We studied the dependence of the free thyroxin (T4) index and the ratio of T4 to thyroxin-binding globulin (TBG) on TBG concentrations, using sera from cases of congenital TBG deficiency and congenital TBG excess. Two such sera with similar concentrations of albumin, transthyretin, and free T4 were mixed to provide test samples with TBG concentrations covering the range of clinical interest without changes in the other T4-binding proteins. Total T4, free T4, TBG, triiodothyronine (T3) uptake, and the free fraction of T3 in serum were measured, and we calculated the free T4 indices, T4/TBG ratios, and the free fraction of T4. A 100-fold variation in TBG concentration was associated with a 10-fold variation in total T4, a fourfold variation in T3 uptake, and a 10-fold variation in the T4/TBG ratio. As TBG concentrations increased, the T4/TBG ratio decreased and the free T4 index increased. The free T4 index did not parallel the T4/TBG ratio, and neither the T4/TBG ratio nor the free T4 index reflected the concentrations of free T4 in serum.     

Hereditary Abnormalities of Thyroxine-Binding Globulin Concentration: A STUDY OF 19 KINDREDS WITH INHERITED INCREASE OR DECREASE OF THYROXINE-BINDING GLOBULIN

Nineteen families with hereditary abnormalities of thyroxine-bindingglobulin (TBG) have been studied, comprising a total of 15 maleswith absent TBG, and 15 females heterozygotic for this condition,and 14 males with excess of TBG and 44 heterozygotic females.Hereditary TBG excess was associated with thyrotoxicosis infour instances, and with myxoedema in three. The typical biochemicalfeatures of TBG abnormality are described, with the clinicalhistories of patients in whom diagnostic and management problemsoccurred.     

Two new inherited defects of the thyroxine-binding globulin (TBG) molecule presenting as partial TBG deficiency.

Serum-denatured TBG (dnTBG) measured in 32 families deficient in native TBG (nTBG) was undetectable in all subjects with complete nTBG deficiency and was high in 2 of 16 families with partial nTBG deficiency. nTBG (in mean micrograms per decaliter +/- SD) in members of the Quebec and Montreal families, respectively were: 258 +/- 54 and 230 in affected men, 747 +/- 190 and 927 +/- 90 in affected women, and 1568 +/- 151 and 1300 +/- 195 in unaffected relatives. Corresponding mean dnTBG levels were: 14.3 +/- 2.9 and 21.3 in affected men, 8.6 +/- 1.0 and 11.6 +/- 3.1 in affected women, and less than 2.1 and less than 2.6 in unaffected relatives. All were euthyroid with normal free thyroxine and thyrotropin levels. In comparison to common type TBG, TBG-Quebec was more heat labile by 10 degrees C and TBG-Montreal by 12 degrees C. The degree of dnTBG elevation and nTBG lability at 37 degrees C were correlated (r = 0.99). Isoelectric focusing showed cathodal shift of all TBG bands: TBG-Quebec by 0.06 isoelectric points (pI) and TBG-Montreal by 0.02 pI. These two TBG variants represent different mutations most likely affecting the polypeptide chain of the molecule. Their inheritance is X-chromosome linked. The instability of these TBGs at 37 degrees C may lead to more rapid degradation in vivo resulting in low nTBG and high dnTBG concentrations in serum.     

Studies on human thyroxine-binding globulin (TBG): I. Purification of TBG and immunologic studies on the relationship between TBG from normal persons and those with TBG “deficiency”

A method for obtaining highly purified thyroxine-binding globulin (TBG) from whole human serum is presented. The method employs relatively simple procedures of step-wise ammonium sulfate precipitation followed by column chromatography on DEAE cellulose and DEAE Sephadex. The final product produces a single protein band on disc electrophoresis. The sedimentation constant of the TBG thus purified is 3.91 and its calculated mol wt is 54,000. An antiserum to the highly purified TBG produced a single arc on immunoelectrophoresis. When the antiserum was reacted against normal human serum or against serum from subjects deficient in TBG, each produced two arcs-one identical with that produced by the antigen alone. The second arc is probably the result of a contaminating protein in the antigen, present in too low a concentration to be detectable by disc gel electrophoresis. It is concluded that some persons with TBG "deficiency" have a circulating protein, immunologically indistinguishable from TBG, which is defective in its ability to bind thyroxine.     

Studies on human thyroxine-binding globulin (TBG). IX. Some physical, chemical, and biological properties of radioiodinated TBG and partially desialylated TBG.

Thyroxine-binding globulin (TBG) and partially desialylated or slow TBG (STBG) were purified from human serum by affinity chromatography. Purified TBG was identical to TBG present in serum by the criteria of electrophoretic mobility, affinity for thyroxine (T4), and heat-inactivation response. Purified STBG had slower electrophoretic mobility and lower affinity for T4. Both bound T4 in an equimolar ratio, were immunoprecipitable, and had similar inactivation t1/2 at 61 degrees C. TBG and STBG were iodinated by the chloramine-T-catalyzed reaction. An average of from 0.02 to 6 atoms I could be incorporated per molecule of the protein by adjusting the conditions of the reaction (time, protein and iodide concentrations). 125-I, 131-I, and 127-I were used. Iodination increased the anodal mobility of TBG but did not affect the reversible T4-binding, precipitation by antiserum, or the heat-inactivation properties. "Heavily" and "lightly" iodinated TBG had identical disappearance half-times from serum in the rabbit. 15 min after the intravenous administration of [131-I]-STBG and [125-I]TBG mixture to rats, more than 90% of the injected 131-I dose was in the liver, and the liver 131-I/125-I ratio was 32-fold that of serum. Selective uptake of STBG by the liver was also observed in the rabbit and in man. The serum [125-I]STBG/[131-I]TBG ratio declined from 1 to 0.2 in 10 min in the intact rabbit but remained unchanged for 1 h in the acutely hepatectomized animal. In the rabbit, t 1/2 was approximately 3 min for STBG and 0.8-3.4 days for TBG. The radioiodine derived from the iodinated proteins is partly excreted in bile but the bulk was precipitable with specific antibodies. Some isotope in the form of iodide appeared in blood and was excreted in the urine. Since radioiodinated TBG and STBG preserve their biologic and immunologic properties they are useful as tracer materials for metabolic studies. In rat, rabbit, and man STBG is rapidly cleared from serum by the liver. Conversion of TBG to STBG may be the limiting step in the regulation of TBG metabolism.     

Effects of Norethandrolone on the Transport and Peripheral Metabolism of Thyroxine in Patients Lacking Thyroxine-Binding Globulin: OBSERVATIONS ON THE PHYSIOLOGICAL ROLE OF THYROXINE-BINDING PREALBUMIN

Studies of the effect of norethandrolone on the transport and peripheral metabolism of thyroxine were carried out in four patients lacking thyroxine-binding globulin. Before norethandrolone administration, values for serum protein-bound iodine (PBI) were decreased (1.8 +/-0.5 mug/100 ml) and the proportion of free thyroxine increased (0.036 +/-0.008%). As a result, values for the absolute concentration of free thyroxine iodine were at the lower end of the normal range (0.63 +/-0.12 mmug/100 ml). During the control thyroxine-turnover study, the thyroxine distribution space was strikingly increased (18.2 +/-7.9 liters) and the fractional rate of thyroxine turnover moderately increased (17.1 +/-11.3%/day), as compared to the expected mean values for normal subjects. Therefore, calculated values for the daily rate of thyroxine clearance were increased even more, ranging between 255 and 500% of normal values. However, owing to the low PBI in these patients, the daily disposal of thyroxine iodine was similar to that expected in normals on the basis of age and weight. During the administration of norethandrolone, the thyroxine-binding capacity of the thyroxine-binding prealbumin increased strikingly in all patients, values averaging 162% of those found during the control period. This increase was associated with a highly significant increase in PBI (133% of control values) and a small but significant decrease in the proportion of free thyroxine, resulting in no significant change in the absolute concentration of free thyroxine iodine. In all four patients, administration of norethandrolone was associated with a pronounced decrease in the thyroxine distribution space to values which averaged 69% of those found during the control period. Values for the fractional rate of thyroxine turnover increased slightly. As a result, thyroxine-clearance rate decreased in all patients. Owing to the reciprocal changes in clearance rate and PBI, no significant change in total daily thyroxine disposal was observed. The present studies reveal that when the thyroxine-binding prealbumin is increased in patients lacking thyroxine-binding globulin, several indices of peripheral thyroxine transport and metabolism are altered. However, these changes were small, even in the absence of thyroxine-binding globulin. It is suggested, therefore, that the effect of changes in thyroxine-binding prealbumin would be even smaller in individuals in whom thyroxine-binding globulin is present.     

Isolation of thyroxine-binding globulin (TBG) by immunoadsorption chromatography: some physical and immunochemical characteristics

Thyroxine-binding globulin (TBG) was isolated from pooled whole human serum by diethylaminoethyl (DEAE) Sephadex anion exchange chromatography followed by immunoadsorption chromatography on a cyanogen bromide-activated Sepharose 4B-sheep anti-human TBG immunoadsorbent. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) of the purified TBG revealed a major protein band with a molecular mass of 65000 and a weak band of molecular mass 54000 in both reducing and non-reducing buffers. Sedimentation velocity analysis revealed an S_20,w coefficient of 4.5S and a calculated molecular mass of 60000. Immunochemical analysis confirmed the purity of the TBG preparation which gave a single precipitin peak on two-dimensional immunoelectrophoresis.     

Methods and clinical evaluation of a new enzyme immunologic method for determination of thyroxin binding globulin (TBG) and T4/TBG quotient: a multicenter study

A heterogeneous enzyme immunoassay for the determination of thyroxine binding globulin (TBG) was developed and assessed in clinical trials in 12 laboratories. The assay is based on the competition principle and employs plastic tubes coated with goat anti-TBG. CV's between 1.4-8.9% for intra-assay precision and 2.9-8.6% for inter-assay precision were found over the concentration range of 4-40 mg/l TBG. In comparative studies using highly purified TBG as standard, values with Enzymun-Test TBG were found to be on average 30% lower than those obtained by various TBG-RIAs. A broad-base study, to determine reference values, was carried out on a group of control persons 18 to 50 years old without previous history of thyroid disease. This study revealed a median of 14.33 mg/l TBG, with 95% of all values between 9.6 and 18.5 mg/l TBG. The median in women of 14.5 mg/l TBG was significantly higher than in men (13.4 mg/l TBG). TBG values in hyperthyroid patients were within the reference range while those in hypothyroid individuals were elevated. Highly elevated TBG values were seen in women receiving oestrogen (median: 22.2 mg/l TBG) and in pregnant women (median: 28.5 mg/l TBG). The T4/TBG ratios made it possible to distinguish between euthyroid, hyperthyroid and hypothyroic subjects (median: 4.9, 11.3 and 1.0, respectively). These ratios were significantly lower in pregnant women (median: 3.1) than in the control persons.     

胎盘球蛋白的制备及体内外的免疫抑制作用

本研究摸索一种简单、方便和高产量的人胎盘免疫球蛋白（placental-eluted gamma globulin,PEGG）的制备方法,探讨其体外对T细胞功能的抑制作用和体内对移植物抗宿主病（GVHD）的预防作用.将胎盘组织残留血洗净,用酸性缓冲液洗脱得到PEGG.用BrdU ELISA检测它对PHA诱导的淋巴细胞增殖和混合淋巴细胞反应（MLR）的影响,用流式细胞仪检测它对T细胞表面CD25,CD69表达的影响,用ELISA方法检测它对T细胞分泌IFN-γ和IL-4的影响.建立小鼠GVHD模型,观察PEGG对小鼠的GVHD临床和病理表现及生存期的作用.结果表明：SDS-PAGE显示PEGG的主要成分是IgG.体外PEGG能有效地抑制PHA诱导的T淋巴细胞的增殖作用和MLR,下调T细胞表面CD25和CD69的表达,抑制IFN-γ产生,促进IL-4的生成.小鼠模型中,PEGG治疗组小鼠生存期较对照组明显延长,且GVHD症状及病理表现较对照组小鼠明显减轻.结论：PEGG在体外能有效地抑制T细胞的增殖、活化,调节Th细胞更多地向Th2细胞方向分化,在体内能有效地预防小鼠GVHD,在治疗GVHD方面具有良好的应用前景.     

Potential Drug Interaction Between Rho(D) Immune Globulin and Live Virus Vaccine

Women often receive Rho(D) immune globulin as well as a live virus vaccine in the immediate postpartum period. The immune globulin product has the potential to interfere with appropriate immune response to the vaccine. Here we describe our approach to identifying and following up on this often overlooked potential drug interaction.     

Alpha(2)-adrenergic receptor signalling in hypertension

Cardiovascular regulation by the sympathetic nervous system is mediated by activation of one or more of the nine known subtypes of the adrenergic receptor family; alpha(1A)-, alpha(1B)-, alpha(1D)-, alpha(2A)-, alpha(2B)-, alpha(2C)-, beta(1)-, beta(2)- and beta(3)-ARs (adrenoceptors). The role of the alpha(2)-AR family has long been known to include presynaptic inhibition of neurotransmitter release, diminished sympathetic efferent traffic, vasodilation and vasoconstriction. This complex response is mediated by one of three subtypes which all uniquely affect blood pressure and blood flow. All three subtypes are present in the brain, kidney, heart and vasculature. However, each differentially influences blood pressure and sympathetic transmission. Activation of alpha(2A)-ARs in cardiovascular control centres of the brain lowers blood pressure and decreases plasma noradrenaline (norepinephrine), activation of peripheral alpha(2B)-ARs causes sodium retention and vasoconstriction, whereas activation of peripheral alpha(2C)-ARs causes cold-induced vasoconstriction. In addition, non-selective agonists elicit endothelium-dependent vasodilation and presynaptic inhibition of noradrenaline release. The evidence that each of these receptor subtypes uniquely participates in cardiovascular control is discussed in this review.     

Clinical Use of Cold Precipitated Antihemophilic Globulin (Factor VIII, CPAG)

A fibrinogen fraction separated from fresh frozen plasma by slow thawing at 2 C contains Factor VIII activity. The administration of this material to six patients with hemophilia A resulted in partial to complete correction of their clotting defect. Clinical hemostasis was associated with these infusions. Normal or higher than normal levels of Factor VIII were observed in one patient given this material in connection with surgery. Side effects did not occur during or after the administration of the fraction. The preparation of this material is simple and can be carried out as a routine procedure in blood banks processing large amounts of fresh frozen plasma.     

The Stability of Factor VIII (Antihemophilic Globulin) in Fresh-Frozen Blood Bank Plasma

Factor VIII (antihemophilic globulin) levels in normal donors varied between 50 and 200 per cent of the mean. The level of Factor VIII in stored fresh-frozen plasma was directly related to the level of the donor and appears likely to be far less influenced by the duration of storage. Collection of blood in plastic bags with the aid of a rocking vacuum device and storage of the plasma at –28 C. for two months resulted in loss of only 10 per cent of Factor VIII activity. Results were definitely inferior when blood was collected by a simple gravity technic. Re-exploration of factors previously shown to influence Factor VIII stability confirmed the fact that temperatures well below freezing and a low pH (such as occurs in ACD plasma) are essential to prevent loss of activity. It is concluded that plasmas with high levels of Factor VIII activity can be obtained best by selecting donors with known high concentrations of this factor.     

Alpha(1) and beta(2) adrenoreceptor agonists inhibit pentylenetetrazole-induced seizures in mice lacking norepinephrine

It has been known for many years that norepinephrine (NE) is a potent endogenous anticonvulsant, yet there is confusion as to which receptor(s) mediate this effect. This is probably due to multiple factors, including the importance of distinct signaling pathways for different seizure paradigms, a lack of comprehensive pharmacological studies, and difficulty in interpreting existing pharmacological results due to the presence of endogenous NE. We sought to circumvent these problems by testing the anticonvulsant activity of selective agonists for most known adrenoreceptors (ARs) in dopamine beta-hydroxylase knockout (Dbh -/-) mice that lack endogenous NE. Dbh -/- mice are hypersensitive to pentylenetetrazole (PTZ)-induced seizures, demonstrating that endogenous NE inhibits PTZ-induced seizures in the wild type. Pretreatment of Dbh -/- mice with an alpha(1)AR or beta(2)AR, but not an alpha(2)AR or beta(1)AR agonist significantly protected against PTZ-induced seizures. In contrast, only the beta(2)AR agonist showed anticonvulsant activity in heterozygous controls. Furthermore, an alpha(1)AR antagonist exacerbated PTZ-induced seizures in control mice, whereas a beta(2)AR antagonist had no effect. We conclude that activation of the alpha(1)AR is primarily responsible for the anticonvulsant activity of endogenous NE in the murine PTZ model of epilepsy. Endogenous NE probably does not activate the beta(2)AR under these conditions, but exogenous activation of the beta(2)AR produces an anticonvulsant effect.