{"paper_id":"ac75a3d7-f519-4e4e-9fcf-9dba084d0c14","body_text":"0X3Xcl \nöndomnO logiea \n\nContents Vol. 92 \nHYPOTHALAMUS \nKalra S. P. Sc Kalra P. S.: Dynamic changes in hypothalamic LH-RH levels as­\nsociated with the ovarian steroid-induced gonadotrophin surge 1 \nMorishita H.y Nakago K., Ii K.. Hashimoto T., Kawatnolo M., Taiiaka T., Higuchi \nK., Miyauchi Y. Sc Ozasa T.: Anovulation and oviductal hyperplasia in rats \ntreated with clomiphene citrate 5 days after birth 577 \nPITUITARY \nBalogh A., Robertson D. M. Sc Diczfalusy E.: Effect of the norethisterone minipill \nin women 428 \nHau man η R. Sc Kühl PL: Interaction of [125I] LH-RH and other oligopeptides with \nplasma membranes of rat anterior pituitaries 228 \nBo/met IL G., Planker J. P., Horowshi R., Wickings E. ]. Sc Schneider H. P. G.: \nSuppression of prolactin secretion by iisuride throughout the menstrual cycle \nand in hyperprolactinaemic menstrual disorders 8 \nCarlson J. C. Si- Kehl S. J.: Prostaglandin-stimulated LH release in cyclic and \novariectomized rats 20 \nde Κ ο/ling J., van Dielen J. Λ. M. /., Tijssen A. M. /. Sc van Rees G. P.: Studies \non a protein synthesis dependent step in LH release by LH-RH 648 \nEversmann T., Fahlbitsch R., Rjosk Η. K. Sc von Werder Κ.: Persisting suppression \nof prolactin secretion after long-term treatment with bromocriptine in patients \nwith prolactinomas 413 \nGolslein /., Vanhaelst L.y Bruno Ο. I). Sc UPI er mite Μ.: Effect of cyproheptadine \non thyrotropin and prolactin secretion in normal man 205 \njaqnes, Jr. S. Sc Gala R. R.: The influence of oestrogen administration in vivo on \nin vitro prolactin release 437 \nKoiter T. /?., Pols-Valkhof N., Zürcher A. F. &• Schuilling G. Α.: LH-secretory \nresponses caused by continuous infusion of LH-RH in pseudoprcgnant rats .... 28 \nLPlermite M., Michaux-Diichenc A. Sc Robyn C.: Tiapride-induced chronic hyper-\nprolactinaemia: interference with the human menstrual cycle 214 \nMarana R., Robertson D. M., Suginami PL S: Diczfalusy E.: The assay of human \nfollicle-stimulating hormone preparations: the choice of a suitable standard .... 599 \nMarana R., Suginami //., Robertson D. M. S: Diczfalusy E.: Influence of the purity \nof the iodinated tracer on the specificity of the radioimmunoassay of human \nfollicle-stimulating hormone 585 \nVII \n\nPeilon F., Philip fron /., Brandl /!. Λ/., Folianno /λ, Lapiane /λ, Dubois Μ. P. 8c \nDecourl J.: Prolactin-sccreting pituitary adenoma in a man with gigantism: \na case report 627 \nPirke Κ. Λ/., Pichler Μ. Μ., Lund R. 8.· Doerr P.: Twenty-four hour sleep-wake \npattern of plasma LH in patients with anorexia nervosa 193 \nRobertson D. M., Pari V., Lindberg Μ. 8c Diczfalusy Ε.: Biologically active lutei­\nnizing hormone (LH) in plasma. V 615 \nSato T. &• Uchigata Y'.: Long-term effects of human growth hormone on plasma \namino acids transport in hypopituitary dwarfism 398 \nSc hams D., Schmidl-Polex B. 8c Kruse V.: Oxytocin determination by radioim­\nmunoassay in cattle 258 \nSchuilling G. Α., Pols-Valkhof N. 8: Koiter T. R.: A possible role of corpora lutea \nwith regard to oestrogen-induced changes in pituitary responsiveness to LH-RH 46 \nSpitz I. M., Trestian S., Cohen H., Arnon N. 8c LeRoith D.: Failure of metoclopra-\nmide to influence LH, FSH and TSH secretion or their responses to releasing \nhormones 640 \nTakano K., Hizuka N., Shizume K. 8c Plasumi Y.: Serum levels of somatomedin A \nand growth during long-term treatment of patients with pituitary dwarfism with \nhuman growth hormone 385 \nvan Buul-Offers S. 8c Van den Brande ]. L.: Effect of growth hormone and pep­\ntide fractions containing somatomedin activity on growth and cartilage met­\nabolism of Snell dwarf mice 242 \nZurate Α., Canales Ε. S., Alger M. 8c Forsbach G.: The effect of pregnancy and \nlactation on pituitary prolactin-sccreting tumours 407 \nTHYROID \nAfrasiabi Α., Valenla L. 8c Gwinup G.: A TSH secreting pituitary tumour causing \nhyperthyroidism: presentation of a case and review of the literature 448 \nBurger A. G., En gier D., Sakoloff C. 8c Stachelt V.: The effects of tetraiodithyro-\nacctic and triiodothyroacetic acids on thyroid function in euthyroid and hyper-\nthyroid subjects 455 \nCarpi Α., Bianchi R., Zucchelli G. C. Del Corso L., Levanti C, Cocci F. Giannessi \nD. 8c Mariani G.: Effect of endogenous thyroid stimulating hormone levels on \nthe secretion of thyroid hormones in man 73 \nCzarnocka B., Nauman /., Adler G. 8c Kielczynski W.: Solubilization and partial \ncharacterization of thyroid membrane TSH binding proteins 512 \nDietrich F. M., Fischer J. A. 8c Bijvoel 0. L. M.: Formation of antibodies to syn­\nthetic human calcitonin during treatment of Pagct's disease 468 \nHaeberli Α., Engler Η., von Grünigen C. Kohler H. 8c Sind er H.: Low molecular \nweight intracellular iodocompounds with long intrathyroidal half-life: remnants \nof thyroglobulin hydrolysis? 105 \nHamada S. 8c Nishimoto M.: Inhibitory effect of certain drugs on thyroid hormone \nbinding by human liver cytosol 277 \nMedeiros-Neto G. Α., Knobcl M., Bronstein Μ. Ζλ, Simoneiii Filho F. F. & \nMailar Ε.: Impaired cyclic-ΛΜΡ response to thyrotropin in congenital hypo­\nthyroidism with thyroglobulin deficiency 62 \nVill \n\nOkamura Κ., I none Κ., Nakashima Τ., Shiroozu A. Sc Yoshinari M.: Iodoamino \nacid synthesis in thyroid lobes in vitro with excellent yield of iodothyronines . . 286 \nSpira Ö., Birkenfeld Α., Gross J. Sc Gordon Α.: TSH synthesis and release in the \nthyroidectomized rat: a) 489 \nSpira O., Birkenfeld Α., Avni Α., Gross J. Sc Gordon Α.: TSH synthesis and release \nin the thyroidectomized rat: b) 502 \nSuzuki H., Kadena Λ7., Takeuchi K. Sc Nakagawa S.: Effects of three-day oral \ncholecystography on serum iodothyronines and TSH concentrations: comparison \nof the effects among some cholecystographic agents and the effects of iopanoic \nacid on the pituitary-thyroid axis 477 \nTheilade P., Molholm Hansen }., Skovsted L. Sc Kampmann ]. P.: Effect of exer­\ncise on thyroid parameters and on metabolic clearance rate of antipyrine in man 271 \nToccafondi R. 5., Rotella C. M., Tanini Α., Fant P. 8: Arcangeli P.: Thyrotrophin-\nresponsive adenylate cyclase activity in thyroid toxic adenoma 658 \nWallace A. L. C, Nancarrow C. D., Evison B. M. Sc Radford Η. M.: The effect of \nthyrotropin releasing hormone on pituitary and thyroid function in pre- and \npost-natal lambs 119 \nWilkin T. /., Gunn Α., Isles T. E.y Crooks J. Sc Swanson Beck ).: The behaviour \nof the thyroidal iodide trap after subtotal thyroidectomy for thyrotoxicosis and \nits implication for the T3-suppression test 85 \nWälinder 0.. Karlsson F. A. Sc Dahlberg P. Α.: Adenyl cyclase activity in human \nthyroid plasma membranes from normal human thyroid tissue and thyroid \nadenomas 95 \nPARATHYROID \nFuss M., Bergans Α., Geurts J., Brauman Pi. Sc Corvilain ).: Effect of rapid vari­\nation of renal function on plasma calcitonin and parathyroid hormone in man . . ISO \nHalse J. Si- Gordcladze ]. 0.: Urinary excretion of calcium, hydroxyproline and \nSVV-cyclic adenosine monophosphate in primary hyperparathyroidism 138 \nWilke R., Harmeyer von Grabe C, Hehrmann R. Sc Hesch R. D.: Regulatory \nhyperparathyroidism in a pig breed with vitamin D dependency rickets 295 \nMAMMARY GLANDS \njorgensen 0. G., Ekeland A. Sc Gautvik Κ. M.: Serum and tissue concentrations \nof immunoreactive calcitonin in patients with breast tumours 522 \nPANCREAS \nBoquist L.: Differences in the blood glucose response of mice to alloxan and al-\nloxan-inhibiting compounds 687 \njärhult J., Ahr en B. Sc Lundquisl I.: Inhibitory effect of somatostatin on insulin \nsecretion during α-adrenergic blockade in three different species 166 \nIX \n\nLenz S., Kühl C, VK«/ig Λ. Molsled-Pedersen L., Qrskov IL Sc Faber 0. K.: The \neffect of ritodrine on carbohydrate and lipid metabolism in normal and diabetic \npregnant women 669 \nSchitsdziarra V,, Rouiller D. Sc linger R. H.: Sympathectomy and prostaglandin \ndeficiency do not prevent gastrogenic hyperglycaemia and hyperinsulinaemia . . 680 \nTurner R. C, Harris E., Ο misted M. Sc Ponsford C: Two abnormalities of glucose-\ninduced insulin secretion: dose-response characteristics and insulin sensitivity . . 148 \nLIVER \nFuruhashi N. Sc Fang V. S.: Sex difference in the induction of lactogenic receptors \nin rat livers 532 \nTakaishi M., Shimizu T. Sc Shishiba Y.: Solubilization of thyroxine-5'-deiodinase \nactivity from rat liver microsome fraction 694 \nADRENALS \nAlvarcz-Buylla R. S: Tsutsumi V.: Adrenocortical function in hypophysectomized \ndogs with parotid gland transplants in direct contact with the basal hypothalamus 710 \nGaillard R. C, Riondel Α., Merkelbach U. & Vallollon M. IL: Changes in plasma \naldosterone following the administration of various combinations of stimuli .... 309 \nSaruta T.. Okuno T., Eguchi T., Nakamura R., Sailo /., Kondo K.. Oka M. Mal-\nsuki S.: Responses of aldosterone-producing adenomas to AGTH and angiotensins 702 \nZachmann M. &• Prader Α.: Unusual heterozygotes of congenital adrenal hyper­\nplasia due to 21-hydroxylase deficiency confirmed by HLA tissue typing 542 \nOVARIES \nLillienberg L., Adlercreutz IL Sc Svanborg Α.: Effect of a sequential oestrogen-\nprogcstin therapy on the plasma level of oestrogens and lipids in post-meno-\npausal women 319 \nUTERUS \nJorgensen J.: The mechanism of the acid activation of rabbit uterine renin 720 \nJorgensen J.: Inactivation of renin in a mixed mitochondrial-lysosomal fraction \nof post-partum uterus 731 \nPREGNANCY \nBelleville F., Lasbennes Α., Nabel, P. &- Paysant P.: HCS regulation in cultured \nplacenta: action of glucose 336 \nχ \n\nKreitmann Β. Sc Bayard F.: Oestrogen and progesterone receptor concentrations in \nhuman endometrium during gestation 547 \nLand B. Sc Seines Α.: Plasma 1,25-dihydroxyvitamin D levels in pregnancy and \nlactation 330 \nReck G., Renner Α., Sinns G. Sc Breckwoldt Μ.: Correlation of plasma non-con­\njugated ocstriol and plasma Cortisol in late human pregnancy 553 \nRigaudiere N.: The androgens in the guinea-pig foetus throughout the embryonic \ndevelopment 174 \nTESTES \nBarbarino A. Sc De Marinis L.: Klinefelter^ syndrome: effects of oestrogen on \ngrowth hormone, prolactin and thyrotrophin release, and on thyrotrophin and \nprolactin responses to thyrotrophin-releasing hormone 347 \nPirke K. M., Krings B. Sc Vogi H.-J.: Further studies on hypothalamic-pituitary-\ntesticular function in old rats 358 \nMISCELLANEOUS \nBecker K. L.y Snider R. //., Moore C. F., Monaghan K. G. Sc Silva Ο. L.: Calcitonin \nin extrathyroidal tissues of man 746 \nBernidz C., Hänsle W. 0., Horn /{., Pickardl C. R., Scriba P. C., Fink E., Kolb PL \nSc Tschesche H.: Isolation, characterization and radioimmunoassay of cortico-\nstcroid-binding globulin (CBG) in human serum - clinical significance and com­\nparison to thyroxine-binding globulin (TBG) 370 \nLarkin L. H., Suarez-Quian C. A. Sc Fields P. Α.: In vitro analysis of antisera to \nrelax in 568 \nPernitclieva-Rostaing Fonlagne J., Adolphe Μ.. Engellnian Ph., Morin P. Sc \nLechat P.: Effect of human chorionic gonadotrophin on phagocytic activity and \nproliferative capacity of rat peritoneal macrophages in culture 187 \nWambach G., Higgins ]. R., Kein D. C. Sc Kaufmann W.: Interaction of synthetic \nprogestagens with renal mineralocorticoid receptors 560 \nXI \n\nACTA ENDOCRINOLOGICA \n92 (1979) 370-384 \nMedizinische Klinik Innenstadt der Universität, \nZiemsscnstraße 1, D-8000 München 2, FRG \nISOLATION, CHARACTERIZATION AND RADIOIMMUNOASSAY \nOF CORTICOSTEROID-BINDING GLOBULIN (CBG) \nIN HUMAN SERUM - CLINICAL SIGNIFICANCE AND COMPARISON \nTO THYROXINE-BINDING GLOBULIN (TBG) \nBy \nC. Bernutz*\\ W. 0. Hänsle, Κ. Horn, C. R. Pickardt, P. C. Scriba, E. Fink**, \nH. Kolb0-) and H. Tschesche*) \nABSTRACT \nIsolation of the corticosteroid-binding globulin CBG was achieved by 5 \nchromatographical steps on Cortisol Sepharose, QAE-Sephadex A-50, Con \nA-Sepharose and hydroxylapatite. The purity of the isolated CBG was \ndemonstrated in Polyacrylamide gel electrophoresis, SDS electrophoresis, \nimmunodiffusion and ultracentrifligation. Microheterogeneity was shown \nin isoelectric focusing by 5 bands in the pH range of 3.7-4.2, which could \nbe reduced to one major band after neuraminidase treatment. The equi-\nmolar binding of Cortisol to CBG was demonstrated by binding studies. \nThe association constant for Cortisol was 2.8 χ ΙΟ8 Μ -1, for progesterone \n!» Abteilung für Klinische Chemie und Klinische Biochemie der Chirurgischen Klinik \nder Universität, Nußbaumstraße 20,. D-8000 München 2, \n-> Forschergruppe Diabetes, Städtisches Krankenhaus München-Schwabing, Kölner \nPlatz, D-8000 München 40, \n•J» Fakultät für Chemie der Universität Bielefeld, Univcrsitätsslraße, D-4800 Bielefeld 1 \nDedicated to Professor Dr. rer. nat. Dr. med. h.c. Theodor Bücher at the occasion of \nhis 65th birthday. \nSupported by Deutsche Forschungsgemeinschaft (SFB 51). \na> Preliminary results were presented in part at the 22nd Symposium of the Deutsche \nGesellschaft für Endokrinologie, Travemünde, 1977. \nC. Β. was awarded with the \"Marius-Tausk\"-Prize of the Deutsche Gesellshaft für \nEndokrinologie 1978 for this work. \nAbbreviations used in this paper: \nCBG, corticosteroid-binding globulin; TBG, thyroxine-binding globulin. \n370 \n\nI.7xlO(5M_1. From analytical ultracentrifugation, the molecular weight \nwas calculated on 50 700; the sedimentation coefficient was 3.6 S, the \npartial specific volume 0.690 ml/g, the Stokes radius 38 Ä and the fric-\ntional coefficient ratio 1.5. \nA sped lie radioimmunoassay for CBG was established using the purified \nCBG for immunization, radioiodination and for calibration standards. The \nnormal range of CBG levels in human serum was 2.4-4.4 mg/100 ml \n(mean ± 2 SD). Studies were performed to compare the levels of CBG and \nthyroxine-binding globulin (TBG). No sex differences but a significant \nbiphasic age dependence were observed for both proteins. In pregnancy \nand under oestrogen treatment of women and men, CBG was demonstrated \nto be the more distinct indicator of oestrogenic activity as compared with \nTBG, whereas the sensitivity of TBG was more pronounced to supposedly \nantiestrogenic substances like Danazol, and in severe disease. No coin­\ncidence of genetic CBG and TBG deficiencies have been found so far. \nIn human serum. Cortisol is bound to a specific transport protein, corticosteroid-\nbinding globulin (CBG) or transcortin, with high affinity and low capacity \nwhich migrates on paper electrophoresis as an alpha1-globulin. In addition \nCortisol is bound to albumin which has low affinity, but high capacity (Daugha-\nday I956a,b; Slaunwhite 8c Sandberg 1959; Slaunwhite et al. 1966; Middoon \n8c Westphal 1967). \nThe concentration of CBG was until now estimated by measuring the total \nbinding capacity of serum for Cortisol using gel filtration and equilibrium \ndialysis {De Moor el al. 1962; Murphy 8c Pattee 1963; Westphal 1971; Schwartz \n8: Hammerslein 1975; Angelt el al. J 977), and recently more specifically by \nradial immunodiffusion {Rosner el al. 1973; Racadot el al. 1974; Van Baelen \n8c De Moor 1974). There is major evidence that CBG has a buffer function for \nthe biologically active free hormone fraction in blood {Slaunwhite et al. 1962; \nDe Moor et al. 1963; Sandberg 8c Slaunwhite 1963), rather than an active \ntransport function for steroid hormones to the target organ cell. But a possible \nrole of proteins in hormone-receptor interactions is also discussed {Westphal \n1971; Werthamer el al. 1973; Wong et al. 1973). The CBG concentrations in \nhuman serum have been estimated mainly under the aspect of oestrogen in­\nfluence {Sandberg 8c Slaunwhite 1959; De Moor el al. 1962; Doe el al. 1964; \nSchwartz 8c Hammer stein 1975) and genetic deficiency variations (Rosner el al. \n1973). \nRecently, characteristic changes of the thyroxine-binding globulin (TBG) \nhave been demonstrated under the influence of different metabolic conditions \n{Horn et al. 1977; Horn 8c Gärtner 1979). Therefore, it was investigated, \nwhether parallelism in changes of these two different transport proteins, CBG \nand TBG could be detected. \nFor this purpose CBG had to be isolated from human serum and a specific \nmethod for its determination had to be established. \n371 \n24 ·•'•• \n\nΜ Α Τ Ε RIALS AND Μ Ε Τ HODS \nReagents. - Pure Cortisol was purchased from the Merck AG, Darmstadt, FRG, [·5Η] \nCortisol (110 mCi/mg Cortisol) from the Radiochemical Center in Amersham, England, \nand Cortisol hemisuccinate from Sigma Chemical Company, St. Louis, USA. Sodium1-r> \niodide (10 Ci/mg 1) came from the Hoechst AG, Frankfurt, FRG. AH-Sepharose 4B \nfor affinity chromatography, Con A-Sepharose, QAE-Sephadex A-50 for ion exchange \nchromatography and Sephadex G-10 were obtained from Pharmacia Fine Chemicals, \nUppsala, Sweden. The hydroxylapatite Biogel HTP was purchased from Bio-Rad \nLaboratories, Richmond, USA. Florisil, acrylamide and bovine gamma globulin were \nobtained from the Serva Biochemica, Heidelberg, FRG. Sodium Lauryl Sulphate (SDS) \nfrom Sigma Chemical Company, St. Louis, USA. Bovine albumin and alpha-methyl-\nD-mannoside from Roth, Karlsruhe, FRG, neuraminidase (Clostridium perfringens) \nfrom Boehringer Mannheim GmbH. The following reagents came from the Merck AG, \nDarmstadt, FRG: N-ethyl-N'-ivS-dimethylaminopropylJ-carbodiimide-hydrochlorid, po-\nlyethylenglycol 6000, complete Freund's adjuvant and all reagents (pro-analysis grade) \nfor the preparation of the buffer solutions. Human transferrin and pertussis vaccine \nwere obtained from the Behringwerke, Marburg, FRG. The human plasma anti-\ncoagulated with acid-citrate-dextrose (ACD) was freshly obtained from the blood bank \nand immediately used for preparation. \nIsoelectric focusing. - Isoelectric focusing studies (Radola Sc Graesslin 1977) of \nisolated CBG were performed in slab gels (16x0.4 cm), using LKB equipment (LKB \nprodukter, Bromma, Sweden). The concentrations of Polyacrylamide and Ampholine® \nsolutions pH 3.5-5.0 and pH 3.5-10 respectively (LKB produkter) were each 5% (v/v). \nAfter isoelectric focusing (500 V, 20 W, 6 hours) gels were either stained using bromo-\nphenol blue or cut in slices of 0.5 cm. After elution with destilled water by diffusion \npH-values were measured with a m.icroelectrode, the CBG-concentrations by radio­\nimmunoassay and [:5H] Cortisol in a /i-scintillation counter. \nIsolation of CBG from human plasma \nAffinity chromatography. - Using a modification of the method described by \nCuatrecasas (1970), 2 g Cortisol hemisuccinate (=4.1 mmol) dissolved in 50 °/o dime-\nthylformamide were covalently bound to 15 g AH-Sepharose 4B (=0.48 mmol amino \ngroups) by activation with 2.17 g carbodiimide (= 10 mmol) for 20 h at room tempera­\nture. The Cortisol Sepharose was then washed alternatively with 1 Μ glycine and 0.2 Μ \nTris-HCl buffer pH 8.6. From 5 liters human plasma the endogenous steroids were \nremoved by 50 g llorml. Then, plasma and Cortisol Sepharose were stirred for 30 min \nat room temperature and for 60 min at 4°C. The plasma was then filtered, the gel was \nwashed with 1000 ml cold 0.2 Μ Tris-HCl buffer pH 8.0 and packed in a water-\njacketed column. From this column CBG was eluted by elevation of the temperature \nup to 30°C with 20 mg Cortisol hemisuccinate in 100 ml 0.16 Μ NaCl in 0.05 Μ Tris-\nHCl buffer pH 8.6. \nPurification of CBG by column chromatography techniques \nThe following purification steps were performed at 4°C. The CBG peak from Cortisol \nSepharose was given on a QAE-Sephadex A-50 column (4.2 χ 24 cm) which was \nequilibrated with 0.18 Μ NaCl in 0.05 Μ Tris-HCl buffer pH 8.6. After washing the \ngel with one column volume of starting buffer, CBG was eluted by the elevation of the \nNaCl concentration up to 0.22 Μ in 0.05 Μ Tris-HCl buffer ρ Η 7.4 in a volume of \n372 \n\n90 ml. This CBG peak was transferred on a Con A-Sepharosc column (3.2x22 cm). \nThe column was washed with one column volume of 0.05 Μ sodium phosphate bulier \npH 7.4 and CBG eluted with 0.06 Μ alpha-methyl-D-mannoside in the same buffer. \nThis CBG peak of 70 ml was dialyzed and concentrated in a collodion bag. The \nfollowing chromatography was performed on a hyclroxylapatite column (1.2x45 cm) \nequilibrated with 0.001 Μ sodium phosphate buffer pH 6.8. The same buffer was used \nfor the elution step. The CBG peak was concentrated and transferred to a QAE-\nSephadex A-50 column (2.2x45 cm), equilibrated with 0.19 Μ NaCl in 0.05 Μ Tris-\nHCl buffer pH 8.6. The final elution was performed with a linear gradient (400 ml) \nfrom 0.19 to 0.24 Μ NaCl in 0.05 Μ Tris-HCl buffer 8.6 in a volume of 75 ml. The \nCBG solution was repeatedly dialyzed in a collodion bag against aqua bidest., frozen, \nlyophilized and stored at - 20°C. The CBG peaks in the eluates from each column were \nidentified using a modification of the [:*H]cortisol-uptake-test on 5 ml Sephadex G-50 \n{Horn et al 1975). \nRadioimmunoassay of CBG \nImmunization. - Rabbits were immunized with 120 ug CBG in complete Freund's \nadjuvant and 0.5 ml pertussis vaccine antigen using a multiple intradermal injection \ntechnique (50 sites) on the back flanks. They were boosted every three weeks with the \nsame technique. \nRadioiodinalion of CBG was done with the chloramine-T-method {Greenwood et al \n1963). The separation of the CBG tracer was performed only on a 1 ml Con A-\nSepharose column. The CBG tracer was eluted with 0.06 Μ alpha-methyl-D-m.annoside \nand diluted in 2 g/1 bovine albumin in 0.05 Μ sodium phosphate buffer pH 7.4. The \ntracer could be used for 8 weeks without further purification. \nFor the radioimmunoassay 100 μ\\ CBG standard solution or 1:100 diluted serum \nwere incubated with 100 μ] CBG tracer and 100 μ\\ diluted CBG antiserum. The \nbound/free-separation could be performed by the polyethylene glycol precipitation \n{Desbuquois Sc Aurbac/i 1971) or the double antibody technique. \nR Ε S U L Τ S \nCriteria of purity and characterization \nElerlrophorclical methods. - The isolated CBG migrated on Polyacrylamide \ngel electrophoresis and SDS electrophoresis {Maurer 1971) each performed in \ntwo different buffer systems pH 8.9 and pH 7.0 and three different gel con­\ncentrations (10, 7.5 and 5%) in one homogeneous band. Immunoelectrophoresis \nand Ouchterlony double diffusion test demonstrated a single precipitation line \nbetween CBG antiserum and CBG solution and normal human serum, respec­\ntively. The overlapping of both precipitation lines indicated identity between \nendogenous and isolated CBG. \nIsoelectric focusing studies revealed a microheterogeneity of 5 different \nbands in the pH range of pH 3.7-4.2. In the same pH area CBG was detected \nin the radioimmunoassay as well as the radioactivity of [3H] Cortisol pre-\nincubated with CBG. Neuraminidase treatment of CBG reduced the micro-\n373 \n\nheterogeneity to one major and one minor band in the pH range of pH 6.0 \n(Fig. 1). \nAnalytical ultracenlrif ligation. — The purity and the homogeneity of the \nisolated CBG could be demonstrated by a homogeneous curve in sedimentation \nvelocity runs and by a straight line in the high speed sedimentation equilibrium \nruns. The sedimentation coefficient corrected to 20° C and water {Schachmann \n1957; Schachmann 8c Edelstein 1966) was calculated to be 3.6 S. Considering \na partial specific volume of 0.690 ml/g derived from the amino acid and \ncarbohydrate composition (Table 1) a molecular weight (Yphantis 1964) of \n50 700 ± 2500 (n = 4) for CBG was determined. The diffusion coefficient was \ncalculated to be 5.6 D, the Stokes radius 38 A and the frictional coefficient \nratio 1.5. \nQuantitative amino acid analysis in two different CBG preparations is sum­\nmarized in Table 1. The high content of aspartic acid and glutamic acid \nexplains the low isoelectric point of CBG. The carbohydrate composition is \nshown in the lower part of Table 1. \npH 5.0 10.0 \nf \npH 3.5 3.5 \nFig. L \nIsoelectric focusing of isolated CBG. Left: untreated CBG, pH gradient 3.5-5.0. Right: \nDesialylated CBG (0.03 U/ml neuraminidase, acetate buffer pH 5.6. 30 min) pH gradient \n3.5-10. \n374 \n\nTable I. \nAmino acid and carbohydrate composition of CBG. The amino acid analysis was \nperformed with two different preparations of CBG, the carbohydrate analysis only \nwith one preparation. Assuming a molecular weight of 50 700 the left column indicates \nthe numbers of amino acid residues per mol CBG, the right column the percentage \nof the single amino acids of the polypeptid residue (molecular weight 32 950). The \nlower part of the table indicates the carbohydrate composition of the second preparation \nof CBG. \namino acids: No. of residues/mol CBG δ­of residue/100 g \nof polypeptid \npreparation I II ι II \nLysine 15 15 6.7 6.7 \nHistidine 11 12 5.2 5.7 \nArginine 10 10 5.3 5.3 \nAspartic acid 35 37 14.1 14.9 \nThreonine 18 17 6.5 6.1 \nSerine 21 16 6.7 5.1 \nGlutamic acid 33 34 14.7 15.2 \nProline - 8 - 2.7 \nGlycine 11 10 2.5 2.2 \nAlanine 14 15 3.7 4.0 \nCystine - - - -\nValine 18 21 6.4 7.4 \nMethionine 12 11 5.4 5.0 \nIsoleucine 15 18 5.9 7.1 \nLeucine 35 39 13.9 15.5 \nTyrosine 13 9 7.1 4.9 \nPhenylalanine 23 25 11.8 12.8 \ncarbohydrate compos tion \nin % by weight \nII \nMannose 9.5 \nGalactose 5.3 \nGlucosamine 10.3 \nSialic acid 10.0 \n375 \n\nBijiding of steroid hormones to the isolated CBG \nThe binding of several steroids to CBG was investigated by two different \nmethods, firstly equilibrium dialysis in micro-cells (Dianorm Apparatus, Dia-\nchemica AG, Switzerland) as the reference method and secondly, gel filtration \non small columns with 2 ml Sephadex G-10 as a very simple method for \nestimation of the relative affinity constants of several steroids. The binding \nstudies were performed in phosphate buffer pH 7.4 at 4° C. The incubation \ntime for gel filtration was 15 min, the dialyzing time 8 h. Equilibrium dialysis \nwas performed for four different steroids using firstly the corresponding tracer \nand secondly [3H] Cortisol in order to test for cross reactivity between the \ndifferent steroids and Cortisol tracer in CBG binding. There was a large \nmeasure of agreement between the calculated binding constants of each method \n(Table 2). Gel filtration resulted in lower association constants than equili­\nbrium dialysis. This was due to the disturbance of equilibrium during the \nfiltration on Sephadex G-10. The binding constant for Cortisol was found to \nbe approximately 8 times lower in gel filtration than in equilibrium dialysis. \nUtilizing this factor for the correction of the association constants of 17a-\nhydroxy-progesterone, 11-deoxyCortisol, progesterone and testosterone, each \ncalculated by displacement of [8H] Cortisol using gel filtration, the corrected \n0.4-\n0,2-\nScatchard Plot \nb= 2.2x108 \nr = 0.97 \nι \n1,5 2,5 bound Cortisol \n[ nM ] \nFig. 2. \nDetermination of the association constant of Cortisol and isolated CBG by means of \nScatchard plot. Ordinate: ratio of CBG bound and free Cortisol tracer. Abscissa: amount \nof bound Cortisol, calculated as product of the percentage of [3H] Cortisol (B/T) and \nthe molecular concentration. \n376 \n\nTable 2. \nAssociation constants of different steroids, determined by equilibrium dialysis using \nthe corresponding tracer for each steroid (first column), equilibrium dialysis using \n[3H] Cortisol tracer only (second column), and gel filtration on Sephadex G-10 using \n[3H]Cortisol tracer only (third column). \nFor methodological details see text. In the right part of the table the molecular dif­\nferences of the investigated steroids compared with Cortisol are indicated. \nassociation constants CM*1] \nmolecular differences compared with Cortisol \n— pregnane structure *-\nsteroid \nequilibrium \ndialysis \ngel \nfiltration \nΔ 1 \ndehydro \nο ο ο \nm \n5 £ \nη?. \nα \nο \nο ο \n\" 5 \nτ. \n•Ι \n5' \n_. > \n:ι \nα \nAndro-\nstene \nstructure \nFluorine \nin 9 oc \nEst rogene \nStructure \nCortisol \nCorrespond irx \nTracer \n2.2 Χ108 \n; Cortisol \nTracer \n2.2x10ö 2.2χ10β \nhigh affinity \nPrednisolone 5.4χ107 \n• \nhigh affinity Corticosterone 5.0Χ107 \n• \nhigh affinity \n17a -Medroxy­\nprogesterone 5.3 χ 107 4.9χ107 28χ107 • • \nhigh affinity \n11 -Deoxy -\nCortisol 1.3 χ 107 1 6 χΙΟ7 2.8χ107 • \nhigh affinity \nDeoxy cortico­\nsterone 2 2χ107 • • \nhigh affinity \nCortisol -\n21 - succinate 1.6 χ 107 • \nmoderate affinity Cortisone 7.2 χ 10* \n• \nmoderate affinity \nMethyl -\nprednisolone Αθχ 106 • • \nmoderate affinity \nProgesterone 17 χ 10β 5.7>. 106 Α.1 χ 106 \n• • • \nmoderate affinity \nPrednisone 3.4χ106 \n• • \nmoderate affinity \nAldosterone 2 6χ 106 \n• • \nmoderate affinity \nTestosterone 2.5 χ 10β 2.9χ10β 16 χ 106 \n• \nlow affinity 9 a - Fluoro -\nhydrocortisone 5.6χ104 • \nlow affinity \nEstriol 2Λχ104 \n• \nlow affinity \nEstradiol 2 3χ104 \n• \nlow affinity \nTriamcinolone -\nacetonid 1.6 χ 10* • \nno affinity Dexamethasone, Ethinylestradiol, Carbenoxolone \nvalues were in the same order of magnitude as found in the equilibrium dia­\nlysis (Table 2). The maximal binding capacity calculated by Scatchard analysis \nwas found to be 715 tug Cortisol per 100 mg CBG indicating an equimolar \nbinding of CBG and Cortisol (Fig. 2). By comparing the association constants \nwith the molecular differences of several steroids to Cortisol (Table 2) it might \nbe supposed that the binding affinity decreased in dependence on the electron \nattraction and the size of the substitute. \n377 \n\nRadioimmunologlial determination of CBG. - The antiserum with the highest \ntiter of 1:160 000, determined by 50% tracer binding, was obtained after the \nthird booster. The maximal tracer binding (specific activity: 31 //Ci///g CBG) \nwas nearly 100%. The nonspecific binding without antiserum in the reaction \nmixture was in the range between 5 to 10%. Using an antiserum dilution of \n1:3000 the limit of detection (3 SD from the zero standard) was 2 ng CBG per \ntube. The 50% intercept was 24 ng CBG per tube, the recovery of added CBG \nstandard in serum was 100%. dilution curves of normal and pregnancy sera \nwere found to be exactly on the calibration curve. The interassay variation \ncoefficient was 7.4%) (mean 3.3 mg/100 ml; η = 23). \nThere was no evidence for cross reactivity of CBG antiserum with alpha.>-\nmacroglobulin, alphao-haptoglobin, alpha!-antitrypsin and albumin in the \nOuchterlony double diffusion test, cross reactivity of TBC* was excluded in the \nradioimmunoassay. Cortisol in serum had no effect on the results of CBG \ndetermination. \nComparison of CBG and TBG levels in human serum \nCBG levels in human serum. - In 40 control persons between the ages of \n15-50 years the range of serum CBG was 2.4-4.4 mg/100 ml. CBG levels of \npatients with Cushing's syndrome (n = 4) and Addison's disease (n = 4) were \nCBG \n(mg /100ml) \nA TBG \n[mg/100 ml] \n<5 <15 15-50 >50 years \nage dependence \nFig. 3. \nAge dependence of CBG and TBG levels. Hatched columns: CBG levels in mg/100 ml. \nOpen columns: TBG levels in mg/100 ml. Hatched lines indicate the normal range \nof CBG and TBG in controls in the age of 15 to 50 years. I mean db so. \n378 \n\nCBG \n[mg/100ml) \n1 TBG \n[mg/100ml]r \nCBG and TBG levels of 4 women subsequently treated with 4 different oestrogen \npreparations. The hatched columns: CBG levels, the open columns: TBG levels. The \nhatched lines represent the normal ranges of CBG and TBG for the age from 15 to 50 \nyears. \nfound in this range. In 134 healthy controls the CBG levels showed a biphasic \nage dependence of CBG levels (Fig. 3). Significant sex differences of CBG \nand TBG levels could not be ascertained in any period of life. \nOestrogen dependence of CBG and TBG. - In pregnancy CBG as well as \nTBG levels increased continuously and reached a plateau during the third \ntrimenon (6.9 ± 0.24 mg/100 ml, η - 16). \nThe effect of exogenous oestrogens on CBG and TBG levels was investigated \nin four ovarectomized women (Fig. 4). The patients were subsequently treated \nwith mestranol (80 //g/day), ethinyl oestradiol (80 //g/day), oestradiol valeri­\nanate (40 mg/14 days), and oestriol (2 mg/day), each preparation for four \nweeks. \nBetween the phases of oestrogen therapy, a period of placebo administration \nwas inserted for 4 weeks. The response of CBG to oestrogens was more \npronounced than the TBG increase. The maximal increase for both was ob­\nserved after mestranol, whereas oestriol had no effect (Fig. 4). In 10 male \n379 \n\npatients undergoing fosfestrol treatment (Honvan®) for a prostatic carcinoma, \na dose-related elevation of CBG levels was observed. Both, CBG and TBG \nlevels reached values of females treated with oestrogens, and again CBG was \nshown to be the more sensitive indicator of oestrogenic activity. \nCBG levels in TBG deficiency stales. - TBG deficiency was induced by \ntreatment with Danazol (2,3-isoxazol-derivative of ethinyl-testosterone, \nWinthrop, Gießen, FRG). Fifteen women were treated with 400 mg Danazol/ \nday for endometriosis, since Danazol is known to induce endometrium atrophy, \nand to suppress ovulation and midcycle peaks of gonadotrophins and oestradiol, \nwhereas the mean basal values of LH and oestradiol remain constant (Goebel \n& Rjosk 1978). The TBG levels were decreased to 50 °/o of the initial values \nafter four weeks of therapy, whereas the CBG levels did not change signi­\nficantly even after 12 weeks (Fig. 5). \nIn 7 patients with severe chronic diseases such as decompensated liver cir­\nrhosis and chronic heart failure, TBG levels were found to be decreased to \n0.72 ± 0.23 mg/100 ml. This was interpreted as symptomatic TBG deficiency. \nIn these patients the mean CBG levels were decreased as well to 2.6 ±1.0 mg/ \n100 ml. But the decrease of CBG was less pronounced than that of TBG. \nIn nine patients (8 men and 1 female) with genetic TBG deficiency (0.4 ± 0.3 \nmg/100 ml, ± SD), CBG levels were found to be in the normal range. \nCBG \n1 mg/100 ml] \ni TBG [mg/100ml) \nmg \nbefore A 8 12 weeks after \nDanazol - therapy \nFig. 5. \nCBG and TBG levels during Danazol therapy. Hatched columns: CBG levels, open \ncolumns: TBG levels. The hatched lines represent the normal range of CBG and TBG \nfor the age from 15 to 50 years. \n380 \n\nDISCUSSION \nThe principle of affinity chromatography as described by Cualrecasas (1970) \nand used at first by Rosncr 8c Bradlow (1971) for the isolation of CBG was \nmodified in our study. \nAlthough Rosncr 8c Bradlow (1971) performed the coupling of Cortisol \nhemisuccinate in pure dioxane and Le Gaillard el al. (1974) thought the coup­\nling in 50% dimethylformamide not to be practicable, we found that the best \nresults were obtained with the latter method. For displacement of CBG from the \nCortisol Sepharose, Cortisol hemisuccinate addition to the elution buffer was \npreferred in order to avoid possible denaturation of CBG which may occur if \nmore aggressive eluents are used. \nThe observation of Rosner 8c Bradlow (1971) who found only CBG and \ngamma globulins in the eluate of the affinity chromatography column could \nnot be confirmed. Therefore, several additional chromatographic purification \nsteps were required. The overall yield of 20% after 5 different preparatory \nsteps was satisfactory. The purity of the isolated CBG was shown by a single \nband in overloaded Polyacrylamide and SDS electrophoreses and by ultra-\ncentrifugation studies. \nThe properties of CBG ascertained in our laboratory agreed for the most \npart with the results of other authors. The molecular weight is reported to be \nin the range of 49 500 to 58 500 {Slaunwhite el al. 1966; Muldoon 8c Westphal \n1967; Le Gaillard et al. 1975). By ultracentrifugation, we determined the value \nof 50 700, the sedimentation coefficient of 3.6 S and the partial specific volume \nof 0.690 ml/g, which corresponds to the values published by Westphal (1971). \nThe hydrodynamic parameters particularly the frictional coefficient ratio sug­\ngest that CBG can still be regarded as a globular protein. The Stokes radius of \nthe molecule is about 38 Ä which corresponded well with other proteins with \na molecular weight in this range. The association constants for Cortisol and for \nother steroids partially obtained by two different methods agreed well with \nthe literature {Westphal 1977; Stroupe el al. 1978). Only the affinity of pro­\ngesterone to CBG was found to be lower {Westphal 1971). \nThe carbohydrate content of 35% by weight and the mean N-acetylneura-\nminic acid content of approximately 16 residues per mol isolated CBG was \nsurprisingly high as compared with the literature {Slaunwhite el al. 1966; Le \nGaillard et al. 1975; Rosner 1976). \nAfter treatment with neuraminidase the microheterogeneity of CBG consist­\ning of 5 single bands was focused to one major band into the alkaline direction. \nThe residual more acidic minor band may be due to incomplete desialylation. \nTherefore the microheterogeneity of CBG may be due only to the different \nN-acetylneuraminic acid content as is already established for other glyco­\nproteins and as recently shown for TBG {Horn 8c Gärtner 1979). \n381 \n\nRadioimniunological quantitation of CBG in scrum. - Although Rosner el al. \n(1973) and Van Baelen Sc De Moor (1974) preferred the subcutaneous or intra­\nmuscular application technique using a ten-times higher amount of CBG, a \nmonospecific antiserum was obtained by the intracutaneous injection of ap­\nproximately 120 //g CBG. As the precision and practicability of the radioim­\nmunoassay was satisfactory, the method was preferable to the radial immuno­\ndiffusion technique (Rosner el al. 1973; Racadol el al. 1974: Van Baelen Sc De \nMoor 1974) for measuring CBG concentrations. \nThe CBG levels in serum of healthy adults corresponded well with the \nvalues estimated by the earlier published methods (Westphal 1971; Rosner et al. \nJ973; Racadol ct al. 1974: Van Baelen Sc De Moor 1974; Rosner 1976). \nCBG levels in normal controls. - Elevated binding capacities of CBG in \nserum have been observed in newborns and infants by several authors (De \nMoor et al. 1962; Angeli el al. 1977; Wagner 1978). These findings could now \nbe ascertained by the direct CBG radioimmunoassay and in addition a further \nincrease of CBG was seen in elder subjects. This biphasic age dependence was \nparallel with the TBG levels (Horn el cd. 1977). Sex differences of CBG or \nTBG levels could not be ascertained in any period of life. These results were \nsurprising with regard to the known oestrogen influence on the levels of both \nproteins, therefore apart from the well known oestrogen induced increase of \nboth transport proteins supposedly other factors have an influence on CBG \nand TBG. \nOestrogen dependence of CBG and TBG levels. - The oestrogen induced in­\ncrease of CBG and TBG levels, well known from earlier investigations (Doe et al. \n1964; Sandberg Sc Slaunwhile 1959; Jngbar 1971; Horn el al. 1977; Wagner 1978). \nwas now ascertained by the quantitative and specific radioimmunological \ndetermination. The investigation of sera in pregnancy and during oestrogen \ntherapy of women and men demonstrated a more pronounced increase of CBG \nas compared with TBG. The increase is probably due to an augmented syn­\nthesis of this protein as the half-life is identical in controls and oestrogen \ntreated persons (Sandberg el al. 1964). After therapy of four women with sup­\nposedly equivalent doses of four different oestrogen preparations, no increase \nof CBG and TBG was found after oestriol. As expected, the increase was \nsignificantly higher after ethinyloestradiol as compared with oestradiol \nvalerianate. Surprisingly the most pronounced increase was induced by me-\nstranol. which may be due to its hepatic metabolism (Bird Sc Clark 1973). \nCBG levels in TBG deficiency stales. - During Danazol therapy, only a \ndecrease of the TBG levels was observed, while the CBG levels did not change. \nDuring the Danazol therapy, clinical signs of peripheral oestrogen deficiency \nwere observed despite of normal oestradiol levels. There is possibly an inter-\n382 \n\nference of Danazol and oestrogens on liver cell receptors, which effects only \nTBG decreases. Likewise, the decrease of TBG levels in catabolic states was \nmore pronounced than that of CBG. These differences in the behaviour of \nCBG and TBG under various hormonal and metabolic influences cannot yet \nbe explained. \nNo coincident genetic defects of the two different transport proteins have \nbeen observed. \nR Ε F ΕR ENCES \nAngeli Α., Frajria R.7 Richiardi L., Agrimonti F. Sc Gaidano G.: Clin. chim. Acta 77 \n(1977) 1. \nBird C. E. Sc Clark A. F.: J. clin. Endocr. S(i (1973) 296. \nCitatrccasas P.: J. biol. Chem. 245 (1970) 3059. \nDaughaday W. H.: J. clin. Invest. 35 (\\956a) 1428. \nDaughaday W. H.: J. clin. Invest. 35 (1956/;) 1434. \nDe Moor P., Deckx R., Raas J. Sc Denef C: Metabolism 12 (1963) 592. \nDe Moor P., Heirwegh K.. Heremans J. F. Sc Declerck-Raskin M.: J. clin. Invest. 41 \n(1962) 816. \nDesbuquois B. Sc Arnbach G. D.: J. clin. 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N., Back N. Sc Sandberg Α. Α.: Science 135 (1962) \n1062. \nSlaunwhile W. R. Sc Sandbcrz Α. Α.: .]. clin. Invest. 3S (1959) 384. \nSlaunwhile W. R. jr., Schneider S., Wissler F. C. Sc Sandberg Α. Α.: Biochemistry ο \n(1966) 3527. \nStroupe S. D., Gray R. D. Sc Westphal U.: FEBS Letters 86 (1978) 61. \nVan Baelen H. Sc De Moor P.: J. clin. Endocr. 39 (1974) 160. \nWagner R. K.: Acta endocr. (Kbh.) Suppl. 218 (1978) 5. \nWerthamer S.. Samuels A. J. Sc Amoral /.; J. biol. Chem. 248 (1973) 6398. \nWestphal U.: Steroid-Protein Interactions. Monographs on Endocrinology. Springer-\nVerlag, Berlin-Heidelberg-New York (1971). \nWestphal U.: Klin. Wschr. 55 (1977) 877. \nWong K. C., Kornel L., Bezkorovainy A. Sc Murphy Β. E. P.: Biochim. biophys. Acta \n(Amst.) 328 (1973) 133. \nYphcmtis D. Α.: Biochemistry 3 (1964) 297. \nReceived on January 18th, 1979. \n384","source_license":"CC0","license_restricted":false}