Monoiodotyrosine Research Articles

Synthesis and Evaluation of Glycosylated Octreotate Analogues Labeled with Radioiodine and 211At via a Tin Precursor

Carbohydration of N-terminus and substitution of a threonine for the threoninol residue at the C-terminus of Tyr3-octreotide (TOC) has resulted in improved pharmacokinetics and tumor targeting of its radioiodinated derivatives. Yet, these peptides are very susceptible to in vivo deiodination due to the similarity of monoiodotyrosine (MIT) to thyroid hormone. The goal of this work was to develop octreotate analogues containing both a sugar moiety and a nontyrosine prosthetic group on which a radioiodine or 211At can be introduced. Solid-phase synthesis and subsequent modifications delivered an iodo standard of the target peptide N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-iodobenzoyl)-Lys0-octreotate (GIBLO) and the corresponding tin precursor N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-[(3-tri-n-butylstannyl)benzoyl]-Lys0-octreotate (GTBLO). GIBLO displaced [125I]TOC from somatostatin receptor subtype 2 (SSTR2)-positive AR42J rat pancreatic tumor cell membranes with an IC50 of 0.46 +/- 0.05 nM suggesting that GIBLO retained affinity to SSTR2. GTBLO was radiohalogenated to [131I]GIBLO and N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-[211At]astatobenzoyl)-Lys0-octreotate ([211At]GABLO) in 21.2 +/- 4.9% and 46.8 +/- 9.5% radiochemical yields, respectively. From a paired-label internalization assay using D341 Med medulloblastoma cells, the maximum specific internalized radioactivity from [131I]GIBLO was 1.78 +/- 0.8% of input dose compared to 9.67 +/- 0.43% for N(alpha)-(1-deoxy-D-fructosyl)-[125I]iodo-Tyr3-octreotate ([125I]I-Gluc-TOCA). Over a 4 h period, the extent of internalization of [131I]GIBLO and [211At]GABLO was similar in this cell line. In D341 Med murine subcutaneous xenografts, the uptake of [125I]I-Gluc-TOCA at 0.5, 1 and 4 h was 21.5 +/- 4.0% ID/g, 18.8 +/- 7.7% ID/g, and 0.9 +/- 0.4% ID/g, respectively. In comparison, these values for [131I]GIBLO were 6.9 +/- 1.2% ID/g, 4.7 +/- 1.4% ID/g, and 0.8 +/- 0.5% ID/g. Both in vitro and in vivo catabolism studies did not suggest the severance of the lys0 along with its appendages from the peptide. Taken together, although GIBLO maintained affinity to SSTR2, its tumor uptake both in vitro and in vivo was substantially lower than that of I-Gluc-TOCA suggesting other factors such as net charge and overall geometry of the peptide may be important.

Iodine species and the endocrine system: thyroid hormone levels in adult Danio rerio and developing Xenopus laevis.

Recently a new approach for the analysis of iodinated organic species in human serum has been developed using liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS). This method enables quantification of iodide, T4 and T3, as well as reverse T3 (rT3) and the synthetic precursors of TH, monoiodotyrosine (MIT), and diiodotyrosine (DIT) in a single injection. In this work, the LC-ICP-MS approach was used to analyze whole-body homogenates of adult male and female zebrafish (Danio rerio) and tadpoles of the African clawed frog (Xenopus laevis) at two different developmental stages (NF58 and 61) according to Nieuwkoop and Faber. The data demonstrate that the LC-ICP-MS method was successful at measuring I-, MIT, DIT, T4, T3, and rT3 in these two species. Furthermore, the method also detected five additional iodinated compounds which are currently unidentified.

Synthesis of 2(S)-benzyl-3-[hydroxy(1?(R)-amino ethyl)phosphinyl]propanoyl-L-3-[125I]-iodotyrosine: a radiolabelled inhibitor of aminopeptidase N

2(S)-benzyl-3-[hydroxy(1′(R)-aminoethyl)phosphinyl]propanoyl-L-3-[125I]-iodo tyrosine was prepared from 1(R)-(N-benzyloxycarbonylamino)ethylphosphinic acid in a six step synthesis. This new iodinated compound, which is a highly efficient aminopeptidase N inhibitor (Ki=0·95 nM), can be used for complete characterization of the biochemical and pharmacological properties of aminopeptidase N and its in vivo inhibition. A high radiochemical purity was obtained with a specific activity of 217 Ci/mmol at the end of the synthesis. Copyright © 2000 John Wiley & Sons, Ltd.

Anionic iodotyrosine residues are required for iodothyronine synthesis.

Biosynthesis of iodothyronines in thyroglobulin occurs by oxidative coupling of two iodotyrosine residues catalyzed by thyroperoxidase. To study the mechanism of iodothyronine formation, iodine-free thyroglobulin was non-enzymatically iodinated and after removal of non-incorporated iodide, incubated with lactoperoxidase and glucose oxidase between pH 4 and 9. The amount of thyroxine (T4). 3,5,3'-tri-iodothyronine (T3), 3,3',5'-tri-iodothyronine (rT3) and 3,3'-di-iodothyronine (T2) formed was measured by radioimmunoassays after hydrolysis of thyroglobulin. T4 is synthesized out of two di-iodotyrosine (DIT) residues in thyroglobulin. The pH dependence of T4 formation fits the dissociation curve of the DIT phenoxy group (pKa 6.5). The formation of T2, synthesized out of two mono-iodotyrosine (MIT) residues, shows a quite different pH dependence. Below pH 6, T2 synthesis could not be observed, while above pH 7.4 a relatively large increase occurred. The values up to pH 8 fitted the dissociation curve of the MIT-phenoxy group with a pKa of 8.7. The gradual loss in enzymatic activity of peroxidase and oxidase in the reaction made the values obtained above pH 8 unreliable. The importance of the ionization of the phenoxy group for the coupling reaction was further consolidated by showing that the pH-dependent oxidation of 2-methoxy-phenol (guaiacol) had 50% maximal product formation at pH 7, a value concordant with pKa 7.0 for the ionization of the phenoxy group of this agent. T3 and rT3 synthesis followed mainly the ionization curve of the inner-ring hydroxyl group, indicating that this ring has the greatest influence on hormonogenesis. Since anion formation facilitates the removal of an electron under oxidative conditions, the pH dependence agrees with the involvement of phenoxy radicals in iodothyronine synthesis, a process that most likely also occurs in vivo since it is mainly T4 that is formed in thyroglobulin.

A micromethod for quantitative determination of iodoamino acids in thyroglobulin.

We describe a new method for quantification of iodoamino acids after enzymatic hydrolysis of thyroglobulin. The procedure involves separation of monoiodotyrosine (MIT), di-iodotyrosine, tri-iodothyronine and thyroxine by reverse phase HPLC with a Vydac C18 stationary phase and a mobile phase of water-acetonitrile-acetic acid. The separation is monitored by sensitive spectrophotometric detection through a 96-well microplate system based on the catalytic Sandell-Kolthoff reaction of iodide on the oxidation of arsenic(III) by cerium(IV). This new microassay is particularly convenient because of its high sensitivity and its rapidity (less than 2 h). It can detect 1 pmol MIT and 0.5 pmol of the other three iodoamino acids with a recovery higher than 96%. Moreover, the 96-well microplate system allows many samples to be tested simultaneously and avoids the use of radiolabeled iodine.

Thyroid peroxidase: kinetics, pH optima and substrate dependency.

The oxidation of iodide, guaiacol and 2,2'-azino-di[3-ethyl-benzthiazoline-(6)-sulphonic acid] and the iodination of tyrosyl residues in bovine serum albumin, catalysed by partly purified thyroid peroxidase, were studied. The enzyme showed pH optima with all electron donors. With the exception of guaiacol, the position of the pH optima depended upon both the electron donor and hydrogen peroxide concentrations. With increased hydrogen peroxide concentrations the optima shifted to lower pH, and with increased iodide concentration to higher pH. For monoiodotyrosine (MIT) formation in bovine serum albumin the position of the pH optimum was also dependent on the hydrogen peroxide concentrations. The position of the pH optimum of the oxidation of guaiacol was pH 9 and independent of substrate and hydrogen peroxide concentrations. It is obvious from these findings that iodination reactions must be studied under well-defined conditions.

Kinetics and mechanism of the peroxidase-catalyzed iodination of tyrosine

The kinetics of iodination of tyrosine by hydrogen peroxide and iodide, catalyzed by both horseradish peroxidase (HRP) and lactoperoxidase (LPO), were studied. The initial rates of formation of both molecular I2 and monoiodotyrosine (MIT) were measured with stopped flow techniques. The following reactions occur in both systems. Enzymatic: FeIII + H2O2-->Fev = O + H2O; Fev = O + I(-)-->FeIII-O-I-; FeIII-O-I- + H(+)-->FeIII + HOI; FeIII-O-I- + I- + H(+)-->FeIII + I2 + HO-. Iodine equilibria: I2 + I-<-->I3-; I2 + H2O<-->HOI + I- + H+. Nonenzymatic iodination, one or both of the following: Tyr + HOI-->MIT + H2O; Tyr + I2-->MIT + I- + H+, where FeIII is native peroxidase, Fev = O is compound I and Tyr is tyrosine. The big difference in the two systems is that the following reaction also occurs with LPO: FeIII-O-I- + Tyr-->MIT + FeIII + HO-, which is the dominant mechanism of iodination for the mammalian enzyme. The overall rate of formation of MIT is about 10 times faster for LPO compared to HRP under comparable conditions. A small decrease in rate occurs when D-tyrosine is substituted for L-tyrosine in the LPO reaction. Thus LPO has a tyrosine binding site near the heme. A kinetically controlled maximum is observed in I3- concentration. Once equilibrium is established, I2 is the dominant form of inorganic iodine in solution. However, hypoiodous acid may be the inorganic iodination reagent.

Carrier-mediated Transport of Monoiodotyrosine Out of Thyroid Cell Lysosomes

Monoiodotyrosine (MIT) crosses the lysosomal membrane of rat FRTL-5 thyroid cells by a carrier-mediated process. In egress studies, MIT lost from inside lysosomes was quantitatively recovered outside lysosomes as MIT, indicating that the compound was transported intact across the lysosomal membrane. In uptake studies, [125I]MIT entry required intact lysosomes and exhibited saturation kinetics. The apparent Km for MIT was approximately 1.5 microM and the Vmax was approximately 0.24 pmol/unit hexosaminidase/min. Countertransport of MIT was demonstrated, with an initial velocity of [125I]MIT uptake which reached a maximum at high intralysosomal MIT loading. Nonradioactive MIT and diiodotyrosine competed to approximately equivalent extents with [125I]MIT for uptake in countertransport experiments. The existence of a lysosomal MIT carrier in thyroid cells may explain how this product of thyroglobulin catabolism is transported to the cytosol for iodine salvage and reutilization.

Studies on the central and peripheral dopaminergic control of PRL secretion in man: failure to demonstrate the existence of a dopamine-mediated prolactin-inhibiting factor.

The existence of a putative central prolactin-inhibiting factor (PIF) distinct from dopamine (DA) but dependent on DA mechanisms for release has been suggested from recent animal studies. We investigated the possibility of the existence of such a PIF in man by combining the use of monoiodotyrosine (MIT), an inhibitor of central DA synthesis, domperidone, a DA receptor antagonist that does not enter the blood-brain barrier and DA itself. 6 normal volunteers underwent three sets of studies: (1) PRL stimulation test to 400 micrograms TRH i.v., 1 g MIT orally or 5 mg domperidone i.v., (2) peripheral DA receptor blockade study in which either domperidone, MIT or TRH was administered at 120 min during a 240-min domperidone infusion (50 micrograms/min) which was preceded by a 5-mg bolus dose of domperidone i.v. and, (3) DA infusion study in which MIT was administered at 120 min during a 240-min infusion of DA in a dose (0.5 microgram/kg X min) known to elevate peripheral DA concentration to levels reported for pituitary portal plasma. In the PRL stimulation tests, the mean +/- SE peak response was significantly greater (p less than 0.002) with domperidone (3,900 +/- 840 mIU/l) than with MIT (1,880 +/- 400 mIU/l) or TRH (2,094 +/- 450 mIU/l). In the peripheral DA receptor blockade study the initial domperidone-induced PRL response was not sustained during the domperidone infusion. Neither a second dose of domperidone nor MIT administration at 120 min resulted in a significant release of PRL.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopaminergic control of gonadotrophin secretion in normal women and in patients with pathological hyperprolactinaemia.

The role of dopaminergic mechanisms in the control of gonadotrophin secretion in normal and hyperprolactinaemic subjects is controversial. Whilst bromocriptine, a potent dopamine agonist, has been used to restore normal gonadotrophin secretion in subjects with pathological hyperprolactinaemia (PHP), dopamine and dopamine agonists have been reported to suppress basal and stimulated gonadotrophin release. We therefore investigated the importance of dopaminergic control of gonadotrophin secretion by studying LH, FSH and PRL responses in normal and PHP subjects to central dopamine synthesis inhibition using monoiodotyrosine (MIT) and to a 4 h dopamine infusion designed to elevate peripheral plasma dopamine concentration to levels reported for pituitary portal plasma (1-6 ng/ml). MIT administration resulted in a significant release of PRL (peak increment 520 +/- 84% above basal) but not of LH or FSH in normal subjects. In PHP subjects there was a blunted PRL response (peak 13.3 +/- 3.5%) to MIT administration and significant LH (P less than 0.05) but not FSH release. Dopamine infusion (0.5 microgram/kg/min) resulted in suppression of PRL (min 19 +/- 3% of basal) but not of LH or FSH. A rebound of PRL (peak 188 +/- 68% of basal) but not LH or FSH occurred on cessation of dopamine. There was an apparent rise in LH (P less than 0.02 vs. normals) but not FSH in PHP patients during dopamine infusion. Plateau dopamine levels achieved during the infusion were 2.9 +/- 0.3 ng/ml and 5.9 +/- 0.8 ng/ml in normal and PHP subjects respectively. The responses to MIT show that dopamine functions as an inhibitor of PRL but not of LH or FSH in normal subjects. In PHP patients the responses suggest increased dopaminergic inhibition of LH release but loss of inhibitory control of PRL release. Physiological concentrations of plasma dopamine do not significantly inhibit LH or FSH release in normal subjects but paradoxically results in an apparent release of LH in PHP patients. We conclude that dopamine mechanisms do not play a significant role in modulating gonadotrophin release in normal subjects. In PHP patients, PRL feedback results in increased hypothalamic dopamine activity which in turn inhibits LH release. We conclude that the inhibitory action of dopamine on PRL release restores LH secretion by removing central dopaminergic inhibition through hypothalamic feedback of PRL.

Prolactin response to dopamine synthesis inhibition using monoiodotyrosine in subjects on oral contraceptives and patients with pathological hyperprolactinemia.

Monoiodotyrosine (MIT), a specific inhibitor of central dopamine synthesis, has been used to study dopaminemediated inhibitory control of PRL release in 10 normal women, 19 women on oral contraceptive steroids (OC), and 14 women with pathological hyperprolactinemia to test the hypothesis that while the mild hyperprolactinemia in OC subjects is under dopaminergic control, there is a loss of inhibitory control in subjects with pathological hyperprolactinemia. Of these latter patients, 6 had normal pituitary fossa on x-rays, and 8 had unequivocal radiological evidence of a pituitary tumor.Subjects on OC had a significantly higher mean basal PRL level (353 ± 34 vs. 280 ± 26 mIU/liter; P 400 mIU/liter; n =...

Divergent phases of responsiveness of thyroid hormone biosynthesis to exogenous thyroid-stimulating hormone in the postnatal rat.

We have found that the thyroids of postnatal rats respond to administration of thyroid-stimulating hormone (TSH) by significant changes in iodine metabolism and hormone biosynthesis. The specific activity of iodine in thyroxine (T4) and 3,5,3′-tri-iodothyronine (T3) was always higher than that in monoiodotyrosine (MIT) and di-iodotyrosine (DIT) in the TSH-treated rats. The highest sensitivity to TSH in this regard was observed during week 2 of life and again at sexual maturity. The lowest thyroid response to TSH with respect to biosynthesis of MIT, DIT, T4 and T3 was observed in weeks 3 and 4 of life rather than in the younger or older rats. However, the specific activities of MIT and DIT were not changed after TSH administration to 2-day-old pups. Increase in thyroid weight and in the specific activity of iodine in the thyroid occurred in almost all of the TSH-treated rats. In contrast, the thyroid iodine content decreased after TSH administration, except in the 2-day-old pups. The specific activity of iodine in the thyroid was highest in the 2-day-old pups and then abruptly declined during week 1 of life and continued to do so until maturity. The thyroid protein content decreased during the first 2 weeks of life but thereafter it was increased in the rats receiving TSH. We conclude that thyroid responsiveness to TSH in terms of thyroid hormone metabolism has several distinct phases in postnatal rats: the first phase, during week 1 of life is characterized by the highest specific activity of iodinated compounds in the thyroid and by the highest percentage representation of T4 and T3; the second, during week 2 of life, by the highest thyroid response to TSH; the third, during weeks 3 and 4 of life, by the lowest thyroid response to TSH; and the last phase in mature rats is characterized by a relatively low specific activity of iodinated compounds in the thyroid but by a good response to TSH stimulation

Bovine parathyroid hormone-(41-84), a hormone fragment with desirable properties for use as radioligand.

Radioiodinated bPTH has been widely used as the labeled ligand in the radioimmunoassay of PTH. We now report the properties of a carboxyterminal fragment of bPTH that has several favorable characteristics when used as radioligand. This peptide, the chief component of a commercial preparation of bPTH, was isolated by gel filtration, where it migrated more slowly than did authentic bPTH-(1-84). It yielded lower nonspecific binding values and more sensitive hPTH assays than were seen with the intact hormone. By immunological criteria this peptide lacked the aminoterminal region of PTH, since hPTH-(1-34) did not inhibit its binding to any of 11 different antisera with known ability to recognize the aminoterminal region of PTH. The peptide did not contain most or all of the carboxyterminal region, however, since its binding to anti-PTH sera was inhibited by hPTH-(44-68) or hPTH-(53-84). Sequential Edman degradation of the iodinated peptide released iodotyrosine at the third cycle, suggesting the structure, bPTH-(41-84). The lower nonspecific binding and enhanced assay sensitivity provided by this peptide suggest that the use of other natural or synthetic fragments of PTH as radioligands might enhance the performance of PTH assays.

Identification of iodotyrosines and some iodothyronines as dimethylaminoazobenzenethiohydantoin derivatives: Application to the sequencing of iodoamino acid containing peptides

The colored Edman's reagent 4- N,N-dimethylaminoazobenzene-4′-isothiocyanate (DABITC), a new coupling agent proposed by J. Y. Chang, E. H. Creaser, and K. W. Bentley (1976, Biochem. J. 153, 607–611) for the microsequence analysis of peptides, proved very convenient for the routine preparation of the dimethylaminoazobenzenethiohydantoin (DABTH) derivatives of monoiodotyrosine (MIT), diiodotyrosine (DIT), thyroxine (T 4), and other iodothyronines including 3,5,3′-triiodothyronine. These derivatives can be separated by the two-dimensional system of thin-layer chromatography used for the common DABTH-amino acids. Application of the coupling method using DABITC to a hormone-containing peptide purified from porcine thyroglobulin gave the following N-terminal sequence: H-Asp- N-Phe-Leu-Leu-Thr-Pro-, with N being Tyr, MIT, DIT, and T 4. The coupling method with DABITC should prove essential in the sequencing of iodoamino acid-containing peptides and in the rapid identification of DABTH-iodoamino acids in different mixtures.

434 SEPARATION AND RADIOIMMUNOASSAY OF TRIIODOTHYRONINE (T3) IN HUMAN BREAST MILK

The purpose of this study was to identify and quantitate T3 in human breast milk (HBM), and to elucidate the relationship of this radioimmunoassayable material with organified 125I in Na125 I-injected rats. The presence of thyroid hormone, and particularly T3, in HBM has been poorly documented, and inconsistency in sample preparation and methods of quantitation have resulted in ambivalent data. Since thyroid hormones are routinely administered per-orally as corrective therapy for hypothyroidism, the presence of T3 in milk has obvious implications for thyroid regulation in the suckling newborn, as the infant will consume ∼ 800 ml/day by 4-6 wks. of age if exclusively breast-fed. Aliquots of either HBM or rat breast milk (RBM) were extracted with acidic ethanol (H+EtOH) after overnight digestion with pancreatin. Samples were eluted from an LH-20 column (Pharmacia) using an ethyl-acetate based solvent mixture. Iodotyrosine and-thyronine standards were run, both in their native form, and after extraction. While the bulk of absorbance (HBM)and organified 125 I (RBM) appears in the monoiodotyrosine (MIT) region of elution profiles, an absorbance peak in HBM in the region of the H+EtOH-extracted T3 standard was observed which corresponds to a radiolabelled peak in RBM. T3-RIA of this region revealed 625 ng/dl in HBM, equivalent to a dose of 5.0 μg/day (800 ml. day consumption in a 1 mo. old infant). We conclude from these data that HBM contains sufficient T3 to be of therapeutic value in amelioration of hypothyroidism in infants.

Restricted food intake and annual thyroid cycle of spotted munia

The spotted munia shows a seasonal cyclicity in total daily food intake as well as in thyroid function. The seasonal decline in thyroid activity is preceded by a 50% reduction in food intake. In January, artificially restricting the food consumption to late summer/fall level (by about 50%; 2.5 g/bird/day over 4 days) led to a significant decrease in conversion of monoiodotyrosine (MIT) to diiodotyrosine (DIT), relative proportions of thyroxine (%T 4) and triiodothyronine (%T 3) in the gland, and plasma protein-bound iodine after the injection of a tracer dose of I at almost all points studied. The plasma radioiodohormone fraction (%T x ), as measured by Sephadex gel filtration, was also significantly less than that in the adlibitum-fed controls at all the points studied. These results were confirmed later (May) by direct measurement (radioimmunoassay) of hormone levels when plasma concentration of total and free T 4 and T 3 were found significantly low in similarly treated birds as compared to controls. A 30% restriction in diet (simulating late spring food intake) did not alter percentage thyroidal 131I uptake, MIT/DIT ratio, glandular %T 4 plasma PBI, and %T x but decreased %T 3 in the gland. A constant fixed restricted diet (2.5 g/bird!day, ambient conditions) over 8 months suppressed the seasonal variation in some thyroid activity parameters (% 131I uptake, % thyroidal T 4 and T 3). The declining ambient temperature failed to restore the thyroid of underfed birds to normal winter efficiency. It is concluded that the seasonal lowering of thyroid activity in spotted munia may be partly due to minimal levels of food intake reached in late summer and fall. The increase in thyroid activity in winter may partly result from an increased food intake in response to the low ambient temperature.

Bile Secretory Apparatus: Evidence for a Vesicular Transport Mechanism for Proteins in the Rat, Using Horseradish Peroxidase and [125I]Insulin

The morphologic mechanisms involved in the uptake, transport, and secretion of proteins into bile were studied in rat liver in vivo. When both horseradish peroxidase (HRP) and insulin were injected into the portal veins of anesthetized rats, these proteins were subsequently detected in bile. Utilizing the technique of combined cytochemistry and quantitative autoradiography, both HRP and [125I]insulin were coincidentally localized within endocytic vesicles within the interior of hepatocytes at various time points after simultaneous intraportal injection. The data suggest that both proteins followed two pathways involving endocytic vesicles of approximately 1000 A in diameter. In the first pathway these protein-containing vesicles were transported through the hepatocyte and subsequently fused with the bile canalicular membrane, resulting in secretion of contained proteins into the biliary space. The second pathway also involved endocytosis into 1000 A vesicles, but these vesicles were transported to the Golgi region and its associated system of lysosomes and endoplasmic reticulum (GERL). Whether the proteins in these vesicles were later secreted into bile was unclear. Measurement of HRP and [125I]insulin or its metabolites, in bile, provided direct evidence that exogenously administered proteins (or their fragments) gain entrance into the biliary space. Studies in which metabolites of [125I]insulin, [125I]monoiodotyrosine (MIT), and 125I, were injected intraportally, demonstrated that less than 10% of [125I]MIT and less than 1.5% of Na125I were retained in perfusion-fixed and processed liver tissue. This study suggests that proteins in blood plasma are taken up by hepatocytes and secreted into bile via a vesicular transport mechanism.

Radio-iodination of plasma membranes of toad bladder epithelium

The present report describes high yield enzymatic radio-iodination of the apical and basal-lateral plasma membranes of toad bladder epithelium, by a procedure that does not breach the functional integrity of the epithelium, as assessed by the basal and vasopressin-sensitive short-circuit current (SCC). Restriction of the label to the membrane surface, was ascertained by light and electron-microscopic autoradiographs. On the apical surface, the grains were over the glycocalyx and the plasma membrane. Analysis of the labeled glycocalyx by agarose gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as enzymatic and pH-dependent hydrolysis indicated that the glycocalyx is a trichloro-acetic acid-soluble macromolecular complex of high molecular weight composed of a peptide moiety attached to large prosthetic groups (presumably carbohydrates) by O-glycosidic bonds. Analysis of the labeled apical plasma membrane components by agarose gel filtration and SDS-PAGE disclosed the presence of six major species of apparent molecular weights: 23,000, 28,000, 37,000, 44,000, 68,000, and 95,000. More than half of the membrane-associated radio-iodine was in two bands of molecular weights 37,000 and 44,000. Concentrations of vasopressin and cyclic AMP sufficient to increase the SCC significantly did not modify the extent of membrane labeling or the distribution of the label among the apical membrane components (presumably proteins) as assessed by SDS-PAGE. Iodination in the presence of amiloride inhibited incorporation but did not change the pattern of the distribution of the label among the components resolved by SDS-PAGE. Iodination of basal-lateral plasma membranes, at a yield comparable to that obtained with apical labeling, was attained after about 30 min of exposure of the intact bladder to the labeling solutions. Approximately 25% of the basal-lateral labeling was lost when the epithelial cells were harvested after collagenase treatment, implying that some iodination of the basement membrane had taken place. Less than 10% of iodination of the apical or basal-lateral surfaces was accounted for by lipid-labeling. Analysis of the labeled apical and basal-lateral species by enzymatic digestion and thin layer chromatography disclosed that virtually all the radioactivity was present as mono-iodotyrosine (MIT).

The effect of dietary ingestion of oxalic acid on thyroid function in male and female Long-Evans rats

Male and female Long-Evans rats revealed decreased body weights and restricted growth rates when placed on a diet of Purina laboratory chow which was supplemented with 2.5 and 5.0% oxalic acid for a period of 70 days. Male and female rats ingesting 5.0% oxalic acid in the diet showed severe losses in body weight. Significant reduction in thyroid weight, 24-hr thyroid uptake of 125I, and plasma radioactivity were observed together with a significant increase in plasma thyroid-stimulating hormone in male and female rats ingesting dietary 5.0% oxalic acid. Chromatographic analysis of thyroid hydrolysates revealed an increase in thyroid labeling of monoiodotyrosine (MIT) which was associated with a decrease in labeling of diiodotyrosine (DIT) leading to elevation in MIT : DIT ratios. While the labeling of tetraiodothyronine (T 4) was unaffected, a significant increase in triiodothyronine (T 3) resulted in elevation of the T 3 : T 4 ratios. These results indicate that chronic ingestion of oxalic acid at the level of 5.0% can lead to a state of hypothyroidism.

The effect of mercuric chloride on thyroid function in the rat

Oral administration of 3 mg of mercuric chloride for 6 consecutive days increases thyroid function as noted by an increase in thyroid 131I release rate. Administration of 2.5 mg of mercuric chloride daily for 40 days enhanced thyroid weight, thyroidal 125I uptake, and serum PB125I. Chromatographic analysis of thyroid pronase hydrolysates of mercury-treated rats displayed a pattern of distribution in thyroid labeling which demonstrated an increased percentage of labeled monoiodotyrosine (MIT), no alteration in percentage of labeled diiodotyrosine (DIT) or T4, but a significant reduction in percentage of labeled T3 indicating perhaps a coupling defect in synthesis of T3 exerted by mercury. Administration of mercuric chloride at 100 ppm for 3 months results in a decrease in thyroidal 131I uptake and depression in the thyroidal 131I release rate which were permanent and irreversible. These findings indicate that the influence of mercuric chloride ingestion on thyroid function probably reflects the quantity of mercuric chloride administered and the length of time during which exposure to mercury persists.