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Biomedical subjects

S Refetoff

Publications and source records attributed to S Refetoff.

At least 145 records · Page 8Linked to original sources
PubMed1989

Sequence of the variant thyroxine-binding globulin of Australian aborigines. Only one of two amino acid replacements is responsible for its altered properties.

A form of thyroxine-binding globulin (TBG) with reduced affinity for hormone and increased susceptibility to heat and acid denaturation has been identified in Australian Aborigines (TBG-A). Results of heat denaturation of TBG established that the TBGA allele is X linked and has a frequency of 50.9% in Western Australian Aborigines. The sequence of an isolated TBGA allele differed at two positions from that of the normal TBG allele (TBGC). One substitution was in codon 191, ACA (threonine) rather than GCA (alanine), and the other was in codon 283, TTT (phenylalanine) instead of TTG (leucine). These nucleotide substitutions resulted in the loss of sites for the enzymes Bgl 1 and Tth 111 II, respectively. The nucleotide substitutions in the TBG-A allele was confirmed by digestion of genomic DNA segments amplified using the polymerase chain reaction. The Bgl 1 and Tth 111 II sites were absent in the genes of two Aboriginal men expressing TBG-A and were present in those of three Aboriginal and six Caucasian males expressing TBG-C. The TBG gene of a seventh Caucasian male possessed the Bgl 1 site but had lost the Tth 111 II site; sequencing of this allele revealed only the substitution in codon 283 identical to that in the TBGA allele. As the biochemical properties of TBGPhe-283 expressed by this individual were indistinguishable from normal TBGLeu-283, we believe that the abnormal properties of TBG-A are due to substitution of alanine for threonine at residue 191.

Adolescent↗
PubMed1989

A new inherited abnormality of thyroxine-binding globulin (TBG-San Diego) with decreased affinity for thyroxine and triiodothyronine.

Evaluation of a family in which males were clinically euthyroid despite having low serum total T4 (TT4) and free T4 index (FT4I) values revealed the presence of a new inherited T4-binding globulin (TBG) variant (TBG-San Diego). Two brothers had low TT4 (39 and 49 nmol/L; normal range, 64-154 nmol/L) and FT4I (4.0 and 4.4; normal range, 6.0-10.5) values, while their grandfather, despite treatment with T4, had a low TT4 (53 nmol/L) and normal FT4I (7.2) in the presence of suppressed TSH (less than 0.1 mU/L). When measured by RIA, the mean TBG concentration (TBG-RIA) of the three affected males was low normal [160 +/- 56 (+/- SD) nmol/L; normal range, 151-253]. Their TBG-binding capacity measured by a T4 binding assay at saturation (TBG-CAP) was similar, giving a mean TBG-RIA/TBG-CAP ratio not significantly different from 1.0. In these males, the TBG affinity for T4 (Ka = 0.48 +/- 0.04 x 10(10) mol-1) was less than that in subjects with the common type TBG (TBG-C; Ka = 1.10 +/- 0.14 x 10(10) mol-1; P less than 0.0001) and similar to that in Aboriginal males from Australia with the variant TBG-A (0.52 +/- 0.10 x 10(10) mol-1). TBG affinity for T3 in the affected males (Ka = 0.68 +/- 0.05 x 10(9) mol-1) was less than that in subjects with TBG-C (1.39 +/- 0.12 x 10(9) mol-1; P less than 0.00001), but greater than that in males with TBG-A (0.44 +/- 0.03 x 10(9) mol-1; P less than 0.0005). The rate of TBG denaturation at 56 C was increased in the affected males (t1/2 = 28.4 and 26.9 min) compared to that in subjects with TBG-C (t1/2 = 54.1 +/- 7.1 min), but was lower than that in males with TBG-A (t1/2 = 20.8 +/- 1.9). A value intermediate between those in affected males and normal subjects was found in serum from an obligatory heterozygote. Microheterogeneity on isoelectric focusing was normal. The exact nature of this structural TBG variant is not yet known. The presence of a TBG mutant should be considered in healthy, clinically euthyroid patients with low serum TT4 and free T4 index values, especially when such low values are found in several members of the same family.

Adult↗
PubMed1989

A mutation causing reduced biological activity and stability of thyroxine-binding globulin probably as a result of abnormal glycosylation of the molecule.

T4-binding globulin (TBG), a 54-kilodalton glycoprotein, is the major thyroid hormone transport protein in man. The exact nature of the mutations causing X chromosome-linked TBG deficiency, which affect about 1 in 2,500 newborn males, is unknown. Here we report the sequence of a unique variant TBG (TBG-Gary) encoding a protein with severely impaired T4 binding as well as decreased stability at 37 C, resulting in its rapid in vivo denaturation. A single nucleotide substitution in the codon for residue 96 of the mature protein replaces isoleucine with asparagine; this replacement creates an additional site for N-linked glycosylation. The anodal shift of TBG-Gary on isoelectric focusing gel electrophoresis suggests that this new site is likely glycosylated. Since glycosylated is required for TBG to assume its correct tertiary structure, but is not subsequently necessary for maintenance of the biological properties or stability of the molecule, we believe that the likely presence of additional carbohydrate probably affects a higher order structure of the molecule and is thus responsible for the reduced stability and hormone binding activity of TBG-Gary (TBGASN-96).

Base Sequence↗
PubMed1989

Sleep deprivation in the rat: IX. Recovery.

Eight rats were subjected to total sleep deprivation, paradoxical sleep deprivation, or high amplitude sleep deprivation until they showed major deprivation-induced changes. Then they were allowed to sleep ad lib. Three rats that had shown the largest temperature declines died within two to six recovery days. During the first 15 days of ad lib sleep, surviving rats showed complete or almost complete reversal of the following deprivation-induced changes: debilitated appearance, lesions on the paws and tail, high energy expenditure, large decreases in peritoneal temperature, high plasma epinephrine and norepinephrine levels, and low thyroxine levels. The most prominent features of recovery sleep in all rats were immediate and large rebounds of paradoxical sleep to far above baseline levels, followed by lesser temporally extended rebounds. Rebounds of high amplitude non-rapid eye movement (NREM) sleep occurred only in some rats and were smaller and less immediate.

Adrenal Cortex Hormones↗
PubMed1988

Sex hormone-binding globulin in the diagnosis of peripheral tissue resistance to thyroid hormone: the value of changes after short term triiodothyronine administration.

Thyroid hormone is one of several factors that modulate the level of sex hormone-binding globulin (SHBG) in serum. SHBG levels are usually elevated in thyrotoxicosis and have been reported to be normal in a few patients with generalized resistance to thyroid hormone (GRTH). This study was designed to determine whether basal serum SHBG levels or the SHBG response to short term T3 administration could be used as an index of thyroid hormone action and thus serve as a test for the evaluation of patients suspected of having peripheral tissue resistance to thyroid hormone. Serum SHBG, total T4, free T4 index (FT4I), total T3, and TSH levels were measured in 21 normal subjects, 28 hypothyroid patients, 20 thyrotoxic patients, and 10 patients with GRTH. Excluding patients with GRTH, serum basal SHBG values were correlated with FT4I values (r = 0.66; P less than 0.0001). Mean SHBG levels in the patients with GRTH [37.6 +/- 16.2 (+/- SD) nmol/L] were not significantly different from those in the normal subjects (35.1 +/- 19.3 nmol/L) or hypothyroid patients (26.3 +/- 17.1 nmol/L), but were significantly lower than those in the thyrotoxic group (64.7 +/- 19.2 nmol/L; P less than 0.001). All 10 patients with GRTH had basal SHBG values in the normal range, but 7 of 20 (35%) thyrotoxic patients also had normal basal SHBG values. T3 was given orally for three sequential 3-day periods at doses of 50, 100, and 200 micrograms daily to 7 normal subjects, 11 hypothyroid and 3 thyrotoxic patients, and all 10 patients with GRTH. The serum SHBG concentration was measured on the last day at each dosage level. During T3 administration, SHBG levels increased in all individuals with normal tissue responsiveness. The increase above the basal value (delta SHBG) at each T3 dose was similar in normal, hypothyroid, and thyrotoxic individuals (non-resistant subjects). After administration of 50 micrograms T3 daily, the mean delta SHBG level was decreased [-2.9 +/- 5.3 (+/- SD) nmol/L] in the resistant patients and increased (4.0 +/- 4.9 nmol/L; P less than 0.005) in the nonresistant subjects. After administration of 100 micrograms T3 daily, the mean delta SHBG was -4.5 +/- 6.8 nmol/L in the resistant patients and 8.6 +/- 5.1 nmol/L (P less than 0.0001) in the nonresistant subjects. Serum SHBG decreased by more than 2 nmol/L in 6 of 10 (60%) resistant patients, but in no nonresistant subject.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1988

Relationship of oligosaccharide modification to the cause of serum thyroxine-binding globulin excess.

T4-binding globulin (TBG), a glycoprotein with four N-glycosyl complex oligosaccharide chains, exhibits sialic acid-dependent microheterogeneity on isoelectric focusing (IEF). Increasing the sialic acid content of TBG increases its anodal IEF mobility and decreases its rate of in vivo degradation, a mechanism for the elevation of serum TBG levels in pregnancy. In this study, the structure of oligosaccharides in TBG from subjects with various conditions associated with TBG excess was determined by measuring the proportion that bound to Concanavalin-A (Con-A). Since oligosaccharides with three or more branches (antennae) attached to the trimannosyl core are excluded from binding to Con-A, the percentage of serum TBG not bound to Con-A (% peak A) represented the portion of TBG molecules with three or more antennae in all oligosaccharide chains interacting with Con-A. Peak A contained the most anodal IEF bands, while the Con-A-bound TBG (peak B) contained the cathodal bands. Serum samples from 10 normal men and 10 premenopausal women did not significantly differ in terms of TBG levels, % peak A, or IEF mobility and were combined as a single group (normal). Eight subjects with elevated serum TBG levels due to inherited TBG excess [62.0 +/- 10.1 (+/- SD) mg/L] or 2 receiving 5-fluorouracil treatment (26.2 and 31.3 mg/L) compared to 20 normal (14.7 +/- 3.3 mg/L) had % peak A values and IEF mobility similar to those in normal subjects. On the other hand, high serum TBG levels in 8 women during the third trimester of pregnancy (39.2 +/- 5.3 mg/L), 2 women taking oral contraceptives (25.7 and 27.0 mg/L), and 3 women with acute hepatitis (34.8 +/- 4.8 mg/L) were associated with significant elevations of % peak A values (5.68 +/- 1.73%, 3.31% and 2.41%, and 3.25 +/- 0.78%, respectively) compared to those in normal subjects (1.33 +/- 0.40%), as well as increased anodal mobility on IEF. Treatment of a man for 3 days with ethinyl estradiol produced similar changes. Using data from densitometry measurements of IEF bands of TBG, the degree of anodal shift was quantitated (anodal index). This index correlated with the % peak A (r = 0.92) in all study subjects. We conclude that increased sites for sialylation, resulting from the increased proportions of triantennary oligosaccharide chains, account for the increased anodal mobility of TBG in hyperestrogenemia and hepatitis. Thus, in these two conditions, a reduced TBG degradation rate resulting from oligosaccharide modification is the likely mechanism of increased serum TBG levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1988

Detection of the thyroxine-binding globulin (TBG) gene in six unrelated families with complete TBG deficiency.

T4-binding globulin (TBG) is a glycoprotein of hepatic origin which transports thyroid hormone in serum. Inherited TBG defects in man are X-chromosome linked and are expressed in hemizygotes as complete deficiency, partial deficiency, or excess. Since TBG is not necessary for thyroid hormone action, affected subjects are healthy. Using DNA probes for human TBG, we searched for restriction fragment length polymorphisms in six affected males belonging to 6 unrelated families with inherited complete TBG deficiency and an equal number of normal males. TBG could not be detected in the serum of any of the TBG-deficient males by a specific and sensitive RIA capable of detecting as little as 5 micrograms TBG/L or 0.031% of the average normal serum TBG concentration. DNA isolated from white blood cells was digested with 11 restriction endonucleases, and the digests were submitted to DNA blot analysis using two cloned TBG-DNA probes which together covered the entire protein coding and the 5'-flanking sequences of the TBG gene. A total of 26 different bands were detected on DNA blots, identifying 18 restriction sites located within the 4.2-kilobase TBG gene, which includes intronic, exonic, and 5'-flanking sequences. This analysis, which sampled 2.3% of the total TBG genome, failed to reveal differences in fragment size among the 6 TBG-deficient and 6 normal males examined. One restriction endonuclease (NcoI) identified normal sequences at the putative promoter region of the gene, and four other endonucleases (TaqI, SstII, MspI, and HpaII) recognized the cytosine-guanine dinucleotide phosphate sequences representing potential mutation hot spots. Although C was methylated at these sites, no C to T (thymidine) transitions were found. These data suggest that large deletions, insertions, or rearrangements of the TBG gene, or mutations at sites of methylated cytosine-guanine dinucleotide phosphate dimers are not common mechanisms for inherited complete TBG deficiency in man.

Cloning, Molecular↗
PubMed1988

Normal cellular uptake of thyroxine from serum of patients with familial dysalbuminemic hyperthyroxinemia or elevated thyroxine-binding globulin.

To determine whether thyroid hormone-binding proteins in serum, particularly albumin, facilitate the transfer of T4 into human tissues, we studied cellular T4 uptake (CT4) by human liver (Hep G2) cells from medium containing serum from subjects with familial dysalbuminemic hyperthyroxinemia (FDH) and acquired and familial T4-binding globulin (TBG) excess and patients with normal T4-binding to albumin and normal TBG concentrations. Serum from nine subjects with FDH whose mean serum total T4 (TT4) concentration was 203 +/- 27 nmol/L were matched for TT4 concentrations with serum from nine subjects with acquired TBG excess (TT4, 201 +/- 23 nmol/L) and nine subjects with thyrotoxicosis and normal TBG concentrations (TT4, 205 +/- 28 nmol/L). The subjects' CT4 results were compared to their serum free T4 concentration, measured by equilibrium dialysis (DT4), and their serum free T4 index (FT4I) value. The mean serum DT4 value for the subjects with FDH (23 +/- 5 fmol/L) and those with TBG excess (23 +/- 3 fmol/L) were normal, whereas it was elevated (44 +/- 9 fmol/L; P less than 0.001) for the thyrotoxic patients with normal TBG concentrations. The mean CT4 value also was normal for the subjects with FDH (37.7 +/- 4.9 fmol/plate) and those with TBG excess (36.6 +/- 4.6 fmol/plate), but was elevated for the thyrotoxic patients (62.3 +/- 11.2 fmol/plate; P less than 0.001). In all three groups studied, the relationship between individual CT4 and DT4 values was similar to that previously found in subjects with no T4-binding protein abnormalities. The mean serum FT4I value was lower for the subjects with acquired TBG excess (111 +/- 22) than for the subjects with FDH (133 +/- 22; P less than 0.05), and it was much higher for the subjects with thyrotoxicosis (221 +/- 31; P less than 0.001). In the subjects with FDH and those with thyrotoxicosis the normal relationship between CT4 and FT4I was maintained, while in the subjects with acquired TBG excess, FT4I values were lower than expected. In seven of the nine subjects with TBG excess, the abnormality was associated with conditions known to increase its sialic acid content: hepatitis (one subject), pregnancy (four subjects), and estrogen therapy (two subjects). The CT4 values were similar in nine subjects with acquired TBG excess (seven pregnant women and two subjects with chronic active hepatitis) and five subjects with familial TBG excess (34.8 +/- 4.3 vs. 34.0 +/- 8.6 fmol/plate, respectively).(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Line↗
PubMed1988

Effect of estrogen on the synthesis and secretion of thyroxine-binding globulin by a human hepatoma cell line, Hep G2.

Hyperestrogenemia in humans increases both the concentration of serum T4-binding globulin (TBG) by 2- to 3-fold and the proportion having anodal mobility on isoelectric focusing (IEF). As TBG is synthesized in the liver, we studied the effect of estrogen on TBG synthesis, secretion, and degradation by cultured human hepatocarcinoma cells (Hep G2). beta-Estradiol in concentrations in the range found in pregnancy (10(-7) M) had no effect on the accumulation of immunoreactive TBG in medium over 4 days. The absence of fetal calf serum or phenol red did not alter these findings. The amount of [35S]TBG accumulated 6 h after addition of [35S]methionine was not influenced by exposure to estrogen or to serum obtained from pregnant women. However, 10(-5) M beta-estradiol suppressed TBG more severely than albumin synthesis (34% vs. 9%). The lack of an estrogen effect on TBG synthesis and secretion was supported by experiments showing no effect of estrogen on the disappearance of TBG added to the medium or the accumulation of cytoplasmic TBG mRNA. The same cultures responded to estrogen by a 10-fold increase in nuclear estrogen receptor binding sites and a 2-fold increase in apolipoprotein CII. As TBG in serum, the rate of heat denaturation was not altered in TBG synthesized by Hep G2 cells in the presence of estrogen. In contrast to the effect on TBG in serum, in Hep G2 cells estrogen did not produce an anodal shift on IEF, or increased its proportion not bound to Concanavalin A, nor reduced its clearance rate when injected into rats. However, even untreated Hep G2 cells synthesized TBG with a larger number of anodal IEF bands and proportion of Concanavalin A excluded material than TBG in pregnancy serum. Results support our hypothesis, based on analysis of TBG in pregnancy, that estrogen-induced serum TBG elevation may not be mediated through an increase in synthesis. The failure to observe estrogen induced changes in oligosaccharide structure does not exclude estrogen responsivity of Hep G2 cells. Such effect could be masked by the marked constitutive increase in number of oligosaccharide chain antennae typical in this and other neoplastic tissues.

Animals↗
PubMed1988

A study on the inheritance of thyroxine-binding globulin (TBG) deficiency from data obtained in 13 families detected by a neonatal screening program.

Thirteen families with thyroxine-binding globulin deficiency, detected through probands screened by the Quebec Network of Genetic Medicine, were investigated. These families were divided into two groups, depending on whether hemizygous males had low or undetectable serum thyroxine-binding globulin levels. Five families belonged to the low (hypo-TBGnemic) type while the remaining 8 families belonged to the absent (a-TBGnemic) type. On pedigree analysis, 10 families satisfied the requirements for an X-linked co-dominant mode of transmission. Three families, 2 a-TBGnemic and 1 hypo-TBGnemic, did not satisfy these requirements and three hypotheses had to be put forward to explain these situations: an autosomal recessive mode of transmission, a spontaneous mutation, or an extreme lyonisation of the defective X. Since the thyroxine-binding globulin gene has been localized to the X-chromosome, the two latter explanations appear to be the most plausible.

Female↗
PubMed1987

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.

Female↗
PubMed1987

Thyroid hormone inhibits fibronectin synthesis by cultured human skin fibroblasts.

Fibronectin (Fn) is a glycoprotein composed of two different subunits, each with a mol wt of approximately 230K. Fn is important for cell adhesion and for maintaining normal cell morphology. Recent in vivo studies suggested that thyroid hormone affects the plasma Fn level in man. The aim of this study was to determine whether T3 regulates the synthesis of Fn in cultured human skin fibroblasts. Skin fibroblasts obtained from seven normal subjects were grown in Minimum Essential Medium supplemented with 10% fetal calf serum. After reaching confluence, the cells were exposed for 3 days to the same medium in which fetal calf serum was replaced by 10% thyroidectomized bovine serum without or with added T3. At the end of this period, the cultures were incubated with [35S]methionine (95 microCi/dish) for 4 h before harvesting. Cell lysates and corresponding media were combined, and 35S incorporation into total protein and Fn was determined by trichloroacetic acid precipitation and immunoprecipitation with antihuman Fn immunoglobulin, respectively. Analysis of the immunoprecipitated material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the 35S activity migrated as a 230K band which was displaced by excess unlabeled Fn. Addition of T3 did not affect trichloroacetic acid-precipitable 35S activity but decreased the 35S activity precipitated with anti-Fn in a dose-dependent manner. While addition of 10(-10) M T3 to the medium had no effect, 35S incorporation into Fn was inhibited by 22.5 +/- 7.4% (mean +/- SD) with 10(-9) M T3 (free T3, 6 X 10(-12) M) and by 31.3 +/- 5.8% with 10(-7) M T3. To assess whether the inhibition of Fn accumulation by T3 was due to suppression of Fn synthesis or enhanced Fn degradation, cells were labeled for 4 h with [35S]methionine and chased for another 4 h with excess unlabeled methionine. T3 had no effect on the rate of decline of [35S]Fn during the chase. We conclude that physiologic T3 concentrations inhibit the synthesis of Fn in normal human fibroblasts. This effect provides a new method to study the action of thyroid hormone which may prove useful in the tissue diagnosis of resistance to thyroid hormone.

Adult↗
PubMed1987

Resistance to thyroid hormone diagnosed by the reduced response of fibroblasts to the triiodothyronine-induced suppression of fibronectin synthesis.

The syndrome of generalized resistance to thyroid hormone (GRTH) is due to a defect at the level of target tissues, but its diagnosis is currently based on indirect tests and cumbersome clinical observations. We recently reported that physiological amounts of T3 inhibit fibronectin (Fn) synthesis by human fibroblasts derived from normal donors. The aim of this study was to assess if this observation could be used for the direct tissue diagnosis of GRTH. Skin fibroblasts from seven normal subjects and seven patients with GRTH were grown to confluence and then exposed for 3 days to medium supplemented with 10% thyroidectomized bovine serum with or without added T3. Cells were then labeled with [35S]methionine for 4 h, and the combined medium and cell lysate was examined for the incorporation of [35S]methionine into trichloroacetic acid and anti-Fn-precipitable material, followed by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 35S activity in the 230K protein was corrected for trichloroacetic acid-precipitable 35S activity. To assess the selectivity of the T3 effect on Fn in fibroblasts from normal subjects compared to GRTH patients, the responses of Fn to dexamethasone and Na butyrate were also determined. Addition of 10(-9) and 10(-7) M T3 to the medium (free T3, 6 X 10(-12) and 2 X 10(-9) M, respectively) reduced Fn synthesis by 13.2 +/- 3.2% (+/- SD) and 14.3 +/- 8.2%, respectively, in fibroblasts from patients with GRTH, an effect significantly less (P less than 0.02 and P less than 0.005) than the 22.2 +/- 7.4% and 30.2 +/- 5.0% reduction that occurred in fibroblasts from normal subjects. With the exception of one patient with GRTH, individual values from each of the two groups did not overlap; the clinical studies suggested that this patient had a milder form of GRTH. In contrast, dexamethasone and sodium butyrate stimulated Fn synthesis by 25.6 +/- 8.7% and 268 +/- 125%, respectively, in fibroblasts from patients with GRTH compared to 27.6 +/- 7.2% and 304 +/- 125% in fibroblasts from normal subjects. The difference of the responses between the two groups was not significant. The results indicate that measurement of the response of Fn to T3 in cultured fibroblasts can be used for the tissue diagnosis of GRTH.

Adolescent↗
PubMed1987

Reduced clearance rate of thyroxine-binding globulin (TBG) with increased sialylation: a mechanism for estrogen-induced elevation of serum TBG concentration.

Hyperestrogenemic states, including pregnancy, cause an increase in serum T4-binding globulin (TBG) concentrations and an increase in the proportion of TBG molecules with greater anodal mobility on isoelectric focusing, indicating greater sialic acid content. The possible causal relationship between the degree of sialylation and accumulation of TBG in serum was explored by measuring the in vivo half-lives (t1/2) of TBGs with different isoelectric points. TBG in unfractionated serum and its major peaks, isolated by chromatofocusing and defined by their isoelectric points on isoelectric focusing were each injected iv into rats. The resulting TBG concentrations, measured by specific RIA in serum samples obtained at intervals after injection, were used for the calculation of the t1/2. TBG in serum from a pregnant woman had a significantly longer t1/2 of 17.2 +/- 1.2 h (mean +/- SD) compared to those of 13.3 +/- 1.5 and 12.9 +/- 0.9 h for TBG in serum from a man and a nonpregnant woman, respectively. TBG peaks II, III, IV, and V, with increasing anodal mobility, had progressively longer t1/2 values of 11, 13, 15, and 33 h, respectively. However, TBG peaks of the same mobility on IEF isolated from serum of pregnant or nonpregnant subjects had similar t1/2 values. Neither the TBG concentration nor estrogen had a direct effect on the rate of TBG clearance. Indeed, the t1/2 of TBG from a subject with inherited TBG excess was not different from that of TBG from a nonpregnant woman or a man. Chronic treatment of rats with estradiol did not alter the rate of clearance of injected human TBG. Finally, the more heavily sialylated anodal bands of purified but unfractionated serum TBG, analyzed by Western blots, survived longer in the circulation of a rat. These results indicate that the rate of in vivo metabolism of TBG is dependent on its sialic acid content. The increased proportion of TBG molecules with higher sialic acid content thus contributes to the increase in the serum TBG concentration in hyperestrogenemic states.

Animals↗
PubMed1986

Nuclear thyroid hormone receptors in cultured human fibroblasts: improved method of isolation, partial characterization, and interaction with chromatin.

In order to characterize the nuclear thyroid hormone receptors in human tissue, an improved method for isolation of nuclei from cultured human fibroblasts was developed. This method provided nuclei with a protein/DNA ratio of 2.8 and recovery of 42%. The purity of nuclei was verified by phase contrast and electron microscopy, which showed normal appearance of chromatin structure. Nuclear binding assay was performed by incubation of whole cells at 37 degrees C or isolated nuclei at 22 degrees C with L-triiodothyronine (T3). In both cases, an affinity constant (Ka) of 2.0-3.0 X 10(10) M-1 and an average binding capacity of 41 femtomoles of T3/100 micrograms DNA (3,100 binding sites/nucleus) were obtained. During incubation of the nuclei, 13% to 16% of receptors that had an identical Ka was released into the medium. Salt extraction recovered 85% to 90% of the receptors, which had a Ka of 4.5 X 10(10) M-1 and the capacity of 0.13 pmol of T3/mg protein. The Ka fo. L-thyroxine (T4) was seven to 18 times lower than that for T3, but the capacity was the same in isolated nuclei, receptors released during incubation of nuclei, and in salt-extracted receptors. Of the iodothyronines examined, affinity for triiodothyroacetic acid was the highest, followed by L-T3, D-T3, L-T4. Isokinetic glycerol gradient analysis revealed that salt-extracted receptors had a sedimentation coefficient of 3.4 S, whereas micrococcal nuclease digested receptors showed two major (6.0 to 6.5 and 12.5 S), and two minor (17 and 19 S) peaks. These results were virtually identical to those obtained with rat liver nuclei analyzed in parallel studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗