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Departments of Pediatrics (N.S.K.), Internal Medicine (G.C.S.), and Obstetrics and Gynecology (J.B.H.), Division of Endocrinology, State University of New York, Brooklyn, New York 11203; Immunochemistry Laboratory (A.F.), Health Science Center at Brooklyn, Brooklyn, New York 11203; and Division of Endocrinology, Department of Pediatrics (N.S.K.), Maimonides Hospital, Brooklyn, New York 11219
Address all correspondence and requests for reprints to: Dr. George C. Schussler, Box 57, 450 Clarkson Avenue, Brooklyn, New York 11203. E-mail: . george.c.schussler-new-york{at}worldnet.att.net
Abstract
Thyroxine-binding globulin, a member of the serine protease inhibitor superfamily of proteins (serpins), releases T4 on cleavage by polymorphonuclear elastase. Such cleavage, previously shown to occur during sepsis and with an exogenous inflammatory stimulus, is now demonstrated in the cord blood of normal babies and appears to be part of a physiological inflammatory response in the newborn. In association with the neonatal TSH surge, thyroxine-binding globulin cleavage is likely to contribute to an increased flux of T4 to neonatal tissues at a time when T4-sensitive morphogenic and biochemical changes are occurring.
THYROXINE-BINDING GLOBULIN (TBG), a noninhibitory member of the serine protease inhibitor (serpin) superfamily (1), is subject to characteristic serpin cleavage through the action of polymorphonuclear leukocyte (PMN) elastase (2, 3, 4, 5). A profound conformational change that is associated with such cleavage (2) releases T4 and accounts for a rapid fall of TBG and T4 concentrations during inflammation (3, 4, 5, 6). This has been interpreted as a mechanism for site-specific delivery of T4 to inflammatory loci. Although the function of such delivery in the expression of thyroid hormone effects remains to be determined, it seems unlikely that release of T4 by cleavage of TBG is restricted to pathological inflammation. We sought evidence of TBG cleavage in the normal newborn because of a report that total PMN elastase (complexed plus free) is approximately 8-fold higher in newborn than adult sera (7). Thus, it might be anticipated that TBG cleavage is accelerated in newborns. This would be relevant to the rate and sites of T4 use at this critical developmental juncture.
Subjects and Methods
Subjects
Cord blood was obtained from eight normal babies immediately after clamping and cutting the cord. All of the babies were delivered vaginally. The babies and their mothers were free of inflammatory disease. Adult sera were obtained with informed consent from healthy volunteers (four men, average age 34 yr; and four women, average age 39 yr). Sera from two patients with sepsis that had previously been found to have the 49-kDa TBG cleavage product (4) were used for comparison. Except for the sepsis sera, which had been frozen, all sera were kept at 4 C after separation. The study was approved by the Institutional Review Board.
Laboratory methods: SDS-PAGE and immunoblotting of sera
Adult and cord sera were subjected to SDS-PAGE with immunoblotting for TBG with the following minor modifications of our previously described method (4). Serum (25 µl) was diluted with 225 µl potassium phosphate buffer (pH 7.1) and added to Affi-Gel blue (Bio-Rad Laboratories, Inc. Hercules, CA). The remainder of the procedure was essentially the same as that previously described, except that the antibody against TBG was lot A0158, obtained from DAKO Corp. (Carpenteria, CA). TBG concentrations were determined by the GammaDab [125I] TBG RIA kit (INCSTAR Corp., Stillwater, MN). T4 and TSH were determined in the clinical immunochemistry laboratory with the AxSym system (Abbott Laboratories, Chicago, IL).
Results
As shown in Table 1
, TBG, T4, and TSH concentrations were significantly higher in cord sera compared with normal adult sera. Immunoblots of SDS-PAGE separations of these sera with TBG antibody are shown in Fig. 1. In the immunoblots of the eight cord sera, an approximately 49-kDa band appears below the 54-kDa band. This lower band corresponds to the TBG cleavage product in the sepsis sera and is absent in the lanes containing normal adult sera.
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This study confirms that cleaved TBG is present in cord blood, where others have previously found increased PMN elastase levels (7). This is the first evidence that such cleavage occurs in the absence of sepsis (4) or an exogenous inflammatory stimulus (8). We suggest that the cleavage of TBG is part of the physiological inflammatory response in the newborn, which is associated with increased concentrations of PMN elastase, IL-1, IL-6, and TNF (7, 9, 10). As previously mentioned, TBG cleavage is likely to be a mechanism for site-specific T4 release. This would be consistent with what seems to be an acute requirement for thyroid hormone at birth, when it is involved in the activation of brown adipose tissue metabolism (11), closure of the patent ductus (12), secretion of surfactant by the type II pneumocyte (13, 14), and the increase in erythrocyte 2,3 diphosphoglycerate (which decreases hemoglobin oxygen affinity; Refs. 15, 16). Cleavage of the high concentrations of TBG present in the newborn (17) should amplify the effect of the concurrent TSH surge (18) on the flux of T4 to the tissues. The importance of thyroid hormone for postembryonic development in man and in most vertebrates is well recognized (19, 20). The hypothesis that increased TBG cleavage accelerates the delivery of thyroid hormone perinatally requires comparison with cleavage in earlier stages of fetal life. Because of the difficulty and potential danger of cordocentesis, we have not attempted to determine TBG cleavage in the blood of the normal fetus before parturition. However, amniotic fluid sampling reveals increased fetal elastase activity at term (21). Consistent with this observation is the report of an exponential rise of fetal leukocyte count from 18–40 wk (22). These findings suggest that the observed TBG cleavage and the associated T4 release at birth are the culmination of rapidly increasing fetal elastase activity in the last trimester. Further support for this hypothesis will require direct determination of TBG cleavage during fetal life, which may be possible in an animal model. Pregnancy is also associated with a maternal inflammatory response (23), and we have evidence of TBG cleavage in maternal serum (24). However, it seems unlikely that the 49-kDa cleavage product observed in cord blood originates in maternal plasma, because, despite interest in the role of TBG in T4 transport to the fetus, there is no evidence that TBG or, for that matter, any other transport protein crosses the placenta.
The significance of TBG in the delivery of hormone to tissues remains speculative. No characteristic abnormalities have thus far been reported in families with hereditary TBG deficiency; however, Skjoldebrand Sparre (25) found low maternal TBG to be associated with spontaneous abortion and major congenital malformations. Because TBG cleavage normally occurs in cord sera, it should be useful to focus on perinatal outcomes in TBG-deficient families.
Acknowledgments
We thank Dr. Henry Anhalt (Division of Pediatric Endocrinology, Department of Pediatrics, Maimonides Medical Center, Brooklyn, NY) for his support.
Footnotes
Abbreviations: PMN, Polymorphonuclear leukocyte; TBG, thyroxine-binding globulin.
Received August 14, 2001.
Accepted March 26, 2002.
References
This article has been cited by other articles:
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  J. Simpson, F. L. R. Williams, C. Delahunty, H. van Toor, S.-Y. Wu, S. A. Ogston, T. J. Visser, R. Hume, and with collaboration from the Scottish Preterm Thyro Serum Thyroid Hormones in Preterm Infants and Relationships to Indices of Severity of Intercurrent Illness J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1271 - 1279. [Abstract] [Full Text] [PDF]
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F. L. R. Williams, J. Simpson, C. Delahunty, S. A. Ogston, J. J. Bongers-Schokking, N. Murphy, H. van Toor, S.-Y. Wu, T. J. Visser, R. Hume, et al. Developmental Trends in Cord and Postpartum Serum Thyroid Hormones in Preterm Infants J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5314 - 5320. [Abstract] [Full Text] [PDF]
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N. Murphy, R. Hume, H. van Toor, T. G. Matthews, S. A. Ogston, S.-Y. Wu, T. J. Visser, and F. L. R. Williams The Hypothalamic-Pituitary-Thyroid Axis in Preterm Infants; Changes in the First 24 Hours of Postnatal Life J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2824 - 2831. [Abstract] [Full Text] [PDF]
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