Ether link cleavage is the major pathway of iodothyronine metabolism in the phagocytosing human leukocyte and also occurs in vivo in the rat.

These studies were performed to test the hypothesis that ether link cleavage (ELC) is an important pathway for the metabolism of thyroxine (T(4)) in the phagocytosing human leukocyte. When tyrosyl ring-labeled [(125)I]T(4)([Tyr(125)I]T(4)) was incubated with phagocytosing leukocytes, 50% of the degraded label was converted into [(125)I]3,5-diiodotyrosine ([(125)I]DIT). Of the remaining [Tyr(125)I]T(4) that was degraded, two-thirds was recovered as [(125)I]-nonextractable iodine ([(125)I]NEI), and one-third as [(125)I]iodide. The production of [(125)I]DIT was not observed when phenolic ring-labeled [(125)I]T(4) ([Phen(125)I]T(4)) was used, although [(125)I]NEI and [(125)I]iodide were produced. None of these iodinated compounds were formed in leukocytes that were not carrying out phagocytosis. The fraction of T(4) degraded by ELC was decreased by the addition of unlabeled T(4) and by preheating the leukocytes, findings which suggested that the process was enzymic in nature. ELC was enhanced by the catalase inhibitor aminotriazole, and was inhibited by the peroxidase inhibitor propylthiouracil, suggesting that the enzyme is a peroxidase and that hydrogen peroxide (H(2)O(2)) is a necessary cofactor in the reaction. To test this hypothesis, studies were performed in several inherited leukocytic disorders. ELC was not observed in the leukocytes of patients with chronic granulomatous disease, in which the respiratory burst that accompanies phagocytosis is absent. ELC was normal in the leukocytes of two subjects homozygous for Swiss-type acatalasemia, and aminotriazole enhanced ELC in these cells to an extent not significantly different from that observed in normal cells. ELC was normal in the leukocytes of a patient with myeloperoxidase deficiency, but could be induced by the incubation of [Tyr(125)I]T(4) with H(2)O(2) and horseradish peroxidase in the absence of leukocytes. The in vivo occurrence of ELC in the rat was confirmed by demonstrating the appearance of [(125)I]DIT in serum from parenterally injected [(125)I]3,5-diiodothyronine, but no [(125)I]DIT was produced when [(125)I]3',5'-diiodothyronine was administered. FROM THESE FINDINGS WE CONCLUDE THE FOLLOWING: (a) ELC is the major pathway for the degradation of T(4) during leukocyte phagocytosis, and accounts for 50% of the disposal of this iodothyronine; (b) the NEI and iodide formed by phagocytosing cells are derived from the degradation of the phenolic and tyrosyl rings of T(4), although ELC per se accounts for only a small fraction of these iodinated products; (c) the process by which ELC occurs is enzymic in nature, and its occurrence requires the presence of the respiratory burst that accompanies phagocytosis; (d) the enzyme responsible for ELC is likely to be a peroxidase, although a clear role for myeloperoxidase as the candidate enzyme remains to be established; (e) iodothyronines are also degraded by ELC in vivo, and the quantitative importance of this pathway in various pathophysiological states requires further investigation.

Conversion of L-thyroxine to triiodothyronine in rat kidney homogenate.

Rat kidney homogenates, in phosphate-EDTA buffer, consistently catalyzed the formation of T3 from added L-thyroxine (T4). The formation of T3 was assessed by both paper chromatography and RIA of T3. Conversion of T4 to T3 appeared to be enzymatic, showing pH and temperature optima (pH 7.0 and 37 C, respectively) and tissue and time dependence. Formation of T3 was unaffected by azide, cyanide, or catalase, nor was it dependent upon oxygen; indeed, under anaerobic conditions conversion of T4 to T3 was enhanced. Dialyzed homogenate retained full activity, and no cofactor requirement was demonstrated. A role of iron and thiol groups in the enzymatic formation of T3 from T4 was suggested by the inhibitory action of iron chelators and thiol-blocking reagents. The capacity of kidney for T3 formation was considerable and increased with increasing T4 concentrations, being approximately 2 nmol/g tissue/h at very high T4 levels. The apparent Km was estimated to be 3 x 10(-6) M. The conversion of T4 to T3 was inhibited by propylthiouracil at micromolar concentrations whereas methimazole, iodide, and lithium salts were without effect. The enzymatic activity of the homogenates was associated with its particulate components, the readily sedimenting fractions corresponding to plasma membranes and mitochondria being most active, and was absent from nuclei and cytosol.

Nuclear triiodothyronine binding in mononuclear leukocytes in normal subjects and obese patients before and after fasting.

We evaluated the maximum binding capacity (MBC) and dissociation constant (Kd) of nuclear T3 receptors (NT3R) in mononuclear cells in normal and obese subjects before and after fasting. Mononuclear cells were isolated by isopycnic centrifugation of 50 ml heparinized blood underlayered with Ficoll-Paque. From 1-5 x 10(6) cells (12-60 microgram DNA) were incubated for 2 h at 37 C in 0.5 ml Ham's F-10 medium with 0.5% bovine serum albumin, 25 mM Hepes buffer (pH 7.4), and six T3 concentrations, each in triplicate (free T3 from 7 x 10(-13) to 5 x 10(-11) and 1.5 x 10(-8) M). Nuclei were isolated from washed cells in sucrose (0.25 M)-Tris (20 mM; pH 7.85)-MgCl2 (1.1 mM) containing 0.5% Triton X-100. Cells took up approximately 5% of the medium T3, and this was not significantly influenced by the T3 concentrations in the medium. About 10% of the cellular T3 was bound to nuclei at 7 x 10(-13) M free T3 in the medium. Nuclear binding in the presence of 1.5 x 10(-8) M free T3 was approximately 20% of that at 7 x 10(-13) M. T4 could compete with T3 for nuclear binding, but it was less than 10% as effective as T3 based on free hormone concentrations. MBC and Kd values of NT3R were the means of two separate determinations on successive days with coefficients of variation of 26% and 28%, respectively. The MBC of NT3R in 9 normal subjects was 2.3 +/- 0.4 (SD) x 10(-15) mol T3/100 microgram DNA, and the apparent Kd was 2.3 +/- 0.5 (SD) x 10(-11) M free T3. For 10 obese subjects, the MBC was 2.5 +/- 0.8 (SD) x 10(-15) mol T3/100 microgram DNA, and the Kd was 2.4 +/- 0.6 (SD) x 10(-11) M free T3. During a 16-day total fast, 7 patients lost 10 +/- 2 kg, and the mean serum T3 decreased by 60%. However, the MBC [2.7 +/- 0.9 vs. 2.4 +/- 0.4 (fasted) x 10(-15) mol/100 microgram DNA] and Kd [2.4 +/- 0.7 vs 2.1 +/- 0.4 (fasted) x 10(-11) M free T3] were not significantly altered. Circulating human mononuclear cells contain limited capacity, high affinity binding sites for T3. We are unable to detect differences in either the MBC or Kd for these sites between lean and obese subjects, nor does fasting alter either of these parameters. If fasting alters NT3R in man, such changes are not detectable in circulating mononuclear cells using these techniques.

Low intrathyroidal iodine concentration in non-endemic human goitres: a consequence rather than a cause of autonomous goitre growth.

Iodine may have an inhibitory and, in some circumstances, a stimulatory effect on thyroid follicular cell growth. Exogenous iodine deficiency causes the growth of endemic goitres and it has been claimed that low intrathyroidal iodine stores stimulate growth. On the other hand, the role of iodine, if any, in regulating the growth of human nodular goitres exposed to an ample supply of iodine has not been studied systematically. Very few data on intrathyroidal iodine concentration in this type of goitre are available. In the present work we have investigated total iodine content in 24 samples from 11 clinically and histomorphologically well-defined fast and autonomously growing human nodular goitres from a non-endemic area. Iodine was fractionated into thyroglobulin-iodine and non-thyroglobulin-iodine. The regional distribution of intrathyroidal iodo-compounds was also assessed in three goitres. Total iodine concentration, as well as its sub-fractions, i.e. thyroglobulin-iodine and non-thyroglobulin-iodine, were significantly lower than in normal thyroids. Furthermore, there was large inter- and intraindividual heterogeneity of all iodo-compounds as well as of thyroglobulin. Total iodine concentration varied by a factor of almost 40 between different goitre samples and by a factor of 20 between samples taken from the same goitre. Total non-thyroglobulin-iodine, the only fraction comprising possible cell growth-regulating iodo-compounds, varied by a factor of > 60 between different goitres and by a factor of > 6 between different samples of the same goitre. The low iodine concentration in all our goitre samples did not differ from values reported in the literature for endemic iodine-deficient goitres. Since all goitres studied here were actively growing while exposed to an ample supply of iodine, iodine shortage cannot be a primary and causal factor for the growth of this type of sporadic goitre. Rather, the low concentration and the large inter- and intraindividual heterogeneity of all iodo-compounds appear to be secondary incidental events well explained by the recently developed concept of autonomous thyroid growth.

Differential expression of IgG Fc binding protein (FcgammaBP) in human normal thyroid tissue, thyroid adenomas and thyroid carcinomas.

The genetic events involved in thyroid carcinogenesis are still incompletely understood. Several rearrangements and mutations of oncogenes have been implicated in the development of thyroid papillary carcinomas, follicular adenomas and carcinomas. However, none of these molecular alterations is suitable either as a general marker for the diagnosis of thyroid carcinomas or to differentiate between thyroid follicular adenomas and carcinomas. In order to identify new genes with altered expression which could serve as such markers, we analyzed RNA from thyroid tumor and normal tissue using a novel technique called restriction-mediated differential display. Several differentially expressed genes were identified, including the gene for IgG Fc binding protein (FcgammaBP). Differential expression of FcgammaBP was confirmed by quantitative real-time RT-PCR. Our experiments showed that IgG Fc binding protein (FcgammaBP) is differentially expressed in normal thyroid tissue, thyroid adenomas and thyroid carcinomas. While the FcgammaBP gene is constitutively expressed in normal thyroid tissue, its expression is significantly increased in follicular thyroid adenomas and significantly decreased in papillary and follicular thyroid carcinomas. Thus, measurement of the expression levels of FcgammaBP in thyroid biopsies might help to make the otherwise difficult distinction between a thyroid follicular adenoma and a follicular carcinoma.

Nuclear triiodothyronine binding sites in rat adipocytes.

Most effects of thyroid hormones appear to be mediated by the binding of triiodothyronine (T3) to nuclear triiodothyronine binding sites (NT3BS). Although thyroid hormones influence adipocyte metabolism, NT3BS have not been described in mature adipocytes yet. This report describes T3 nuclear binding in isolated nuclei from rat epididymal fat pad adipocytes. Nuclei were isolated by exposing collagenase-dispersed adipocytes to STM (sucrose, 0.25 mol/L; TRIS, 20 mmol/L; MgCl2, 1.1 mmol/L, pH 7.85) containing 0.5% (vol/vol) Triton X-100. Incubation of nuclei suspended in STM/EDTA (2 mmol/L)/DTT (5 mmol/L) with 125I-T3 and varying concentrations of unlabeled T3 at 37 degrees C for one hour revealed the presence of high-affinity, low-capacity NT3BS. Their MBC was 0.39 +/- 0.04 (SD) ng of T3 per milligram of DNA and their Kd was 1.4 +/- 0.5 (SD) X 10(-10) mol/L T3. Specific binding reached a plateau between 30 minutes and two hours of incubation. The addition of 5 X 10(-7) mol/L T3 to nuclei incubated for one hour with 2 X 10(-11) mol/L T3 completely displaced the specifically bound 125I-T3 within 30 minutes. Thyroxine (T4) and 3, 3', 5'-triiodothyronine (rT3) could displace 125I-T3 from the NT3BS but were less than 10% and 1% as effective, respectively, as T3. Rat epididymal fat pad adipocytes contain NT3BS, the binding characteristics of which are similar to those of rat hepatic NT3BS.

Suprasellar germinomas in childhood and adolescence: diagnostic pitfalls.

The clinical and neuro-endocrine data of seven young male patients with suprasellar germinomas seen between 1984 and 1992 are reported. The most common initial symptom was 'idiopathic' central diabetes insipidus (DI), which occurred in all seven patients. The time interval between the appearance of this first clinical sign and the definitive diagnosis of a suprasellar germinoma ranged from 3 to 66 months. Raised prolactin levels and growth hormone deficiency were indicators of a process located in the hypothalamic-pituitary region. An increased beta-HCG level in the serum or the CSF confirmed the diagnostic suspicion of a germinoma and was helpful as a tumor marker in follow-up. Neuro-radiologic studies (CT or MRI) were also disappointing in the early stage when patients presented only with DI. Later on, as patients developed additional symptoms or signs related to the tumor, imaging studies were positive. Given the variable rate of tumor progression, the nonspecific early signs of hypothalamic-pituitary dysfunction (DI) as well as the often negative early imaging studies, the diagnosis of suprasellar germinoma is difficult but should always be considered in the presence of so-called 'idiopathic' central DI. Repeated brain MRIs are mandatory in young patients with idiopathic DI in order not to miss an underlying suprasellar germinoma.

Granulocyte colony-stimulating factor inhibits neutrophil apoptosis at the local site after severe head and thoracic injury.

BACKGROUND: Tissue injury from mechanical trauma often leads to secondary organ failure. Local accumulation of neutrophils and excessive release of toxic metabolites through inhibition of neutrophil apoptosis may be responsible for capillary leakage and irreversible damage of resident cells of injured tissues. METHODS: The purpose of this study was to examine the presence of apoptosis-inhibiting factors at the local site of tissue injury. Cerebrospinal fluid (CSF) from patients with severe head injury (n = 10; Abbreviated Injury Scale score, 4.5 +/- 0.2 points) and bronchoalveolar lavage fluid (BALF) from patients with serious chest trauma (n = 10; Abbreviated Injury Scale score, 4.1 +/- 0.1 points) were collected on days 1 and 3 after injury and compared with CSF (n = 5) and BALF (n = 16) obtained from patients undergoing elective orthopedic surgery. Neutrophils from healthy humans were incubated with 10% of CSF or BALF for 16 hours. Neutrophil apoptosis was determined by flow cytometric analysis of propidium iodide nuclear staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and May-Grunwald-Giemsa staining. Levels of granulocyte colony-stimulating factor (G-CSF) in CSF and BALF were measured with enzyme-linked immunosorbent assay. RESULTS: CSF and BALF from injured patients significantly inhibited spontaneous neutrophil apoptosis of healthy humans compared with control samples, whereas respiratory burst activity was enhanced (p < 0.05). Moreover, CSF and BALF from injured patients contained increased (p < 0.05) amounts of G-CSF. Neutralization of G-CSF in CSF and BALF from injured patients using monoclonal anti-G-CSF antibody markedly (p < 0.05) reduced the apoptosis-inhibiting effect of those body fluids and decreased the respiratory burst. CONCLUSION: In patients with severe head or chest injury, G-CSF acts locally as a strong inhibitor of spontaneous neutrophil apoptosis, which may cause an increased destructive potential of neutrophils present in injured tissues.