Deiodination Product Research Articles

Quality Assurance Investigations and Impurity Characterization during Upscaling of [177Lu]Lu-PSMAI&T

[177Lu]Lu-PSMAI&T is widely used for the radioligand therapy of metastatic castration-resistant prostate cancer (mCRPC). Since this kind of therapy has gained a large momentum in recent years, an upscaled production process yielding multiple patient doses in one batch has been developed. During upscaling, the established production method as well as the HPLC quality control were challenged. A major finding was a correlation between the specific activity and the formation of a pre-peak, presumably caused by radiolysis. Hence, nonradioactive reference standards were irradiated with an X-ray source and the formed pre-peak was subsequently identified as a deiodination product by UPLC-MS. To confirm the occurrence of the same deiodinated side product in the routine batch, a customized deiodinated precursor was radiolabeled and analyzed with the same HPLC setup, revealing an identical retention time to the pre-peak in the formerly synthesized routine batches. Additionally, further cyclization products of [177Lu]Lu-PSMAI&T were identified as major contributors to radiochemical impurities. The comparison of two HPLC methods showed the likelihood of the overestimation of the radiochemical purity during the synthesis of [177Lu]Lu-PSMAI&T. Finally, a prospective cost reduction through an optimization of the production process was shown.

Endogenous 3-Iodothyronamine (T1AM): More Than We Bargained For

In 2004, the discovery of a new biogenic amine called 3-iodothyronamine (3-T1AM, or simply T1AM) that possessed interesting biological activity was reported (1). Theoretically a decarboxylation and deiodination product of T4, T1AM was extracted from rodent tissue and blood and was identified using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) against authentic synthetic molecular standards. Rodents treated with high single doses of T1AM display a rapidly induced behavioral anergia, hypothermia, reduction in cardiac drive, and hyperglycemia, the mechanism of which involves central nervous system processes (2–4). These hypometabolic actions of T1AM suggest a potential use in ischemic disorders and injuries, and indeed T1AM treatment is neuroprotective in a mouse stroke model (5). Siberian hamsters, a hibernating rodent species, rapidly switch fuel utilization upon T1AM treatment from carbohydrate to pure lipid burning (6). This dramatic effect of single-dose T1AM treatment is sustained for several days and results in selective loss of fat mass and reduction in body weight, suggesting that T1AM may play an important role in regulating energy utilization and metabolism. It has been noted that many of the biological actions of excess T1AM run in the opposite direction of excess T3, or hyperthyroidism, and this and its possible biogenesis from thyroid hormone encourages speculation that T1AM may represent a new and underappreciated arm of the thyroid hormone endocrine axis (7). Since the discovery of T1AM, a prevailing question has been what are the endogenous levels of this putative thyroid hormone metabolite in circulation and tissues? The central importance of this question toward developing a clear understanding of the physiological capacity of T1AM is undeniable; assorted efforts to quantify this substance from tissue and serum samples have been under way for the past 6 yr, and these efforts have met with, at best, limited success (8, 9). Why has the quantitative analysis of what looks on the surface like a fairly ordinary biogenic amine been such a challenge? Research over the past 6 yr has established that T1AM possesses biological properties unlike any other biogenic amine, including an exceptionally long circulating half-life and a tenacious affinity for a serum factor recently identified as apolipoprotein B-100 containing lipoprotein particles (Roy, G., B. Hettinger, T. S. Scanlan, unpublished results). These unanticipated biological properties of T1AM have likely hampered efforts to develop a reliable method of quantitative analysis. However, the past year has brought important new developments— namely two different, yet complementary methods for quantitative assay of endogenous T1AM. The first advance appeared in October, 2010, from Saba et al. (10) at the University of Pisa who reported levels of T1AM in rat tissue and blood as measured by LC/ MS/MS after chemical extraction, and these levels were compared with those of T3 and T4 extracted and quantified by the same method. This approach revealed that rat serum contained 0.30 nM T1AM—a level more than 150 times lower than total T4 and about five times lower than total circulating T3. A few human serum samples were also evaluated with this method and showed similar subnanomolar T1AM concentrations. In contrast, tissue T1AM levels in rat liver, heart, kidney, muscle, and other tissues exceeded T4 and T3 tissue levels by 3to 10-fold depending on the tissue. The principle finding of low circulating and high tissue-associated T1AM concentration suggested that T1AM was either being produced in tissues or accumu-

Transformations of 4,4,5,5,6,6,7,7,7-nonafluoro-2-iodoheptyl glycidyl ether upon the action of nucleophiles and reducing agents

Selective substitution of the iodine atom for hydrogen in 4,4,5,5,6,6,7,7,7-nonafluoro-2-iodoheptyl glycidyl ether has been performed upon the action of Bu3SnH/Bz2O2, which led to the corresponding deiodination product in 82% yield. The epoxide ring opening in the title substrate was observed in its reaction with methanol and acetone in the presence of a Lewis acid, as well as upon the action of thiourea, morpholine, and sodium azide. In the last two cases, the formation of secondary alcohols is accompanied by dehydroiodination.

Thyronamines Are Isozyme-Specific Substrates of Deiodinases

3-Iodothyronamine (3-T 1 AM) and thyronamine (T AM) are novel endogenous signaling molecules that exhibit great structural similarity to thyroid hormones but apparently antagonize classical thyroid hormone (T(3)) actions. Their proposed biosynthesis from thyroid hormones would require decarboxylation and more or less extensive deiodination. Deiodinases (Dio1, Dio2, and Dio3) catalyze the removal of iodine from their substrates. Because a role of deiodinases in thyronamine biosynthesis requires their ability to accept thyronamines as substrates, we investigated whether thyronamines are converted by deiodinases. Thyronamines were incubated with isozyme-specific deiodinase preparations. Deiodination products were analyzed using a newly established method applying liquid chromatography and tandem mass spectrometry (LC-MS/MS). Phenolic ring deiodinations of 3,3',5'-triiodothyronamine (rT3AM), 3',5'-diiodothyronamine (3',5'-T2AM), and 3,3'-diiodothyronamine (3,3'-T2AM) as well as tyrosyl ring deiodinations of 3,5,3'-triiodothyronamine (T3AM) and 3,5-diiodothyronamine (3,5-T2AM) were observed with Dio1. These reactions were completely inhibited by the Dio1-specific inhibitor 6n-propyl-2-thiouracil (PTU). Dio2 containing preparations also deiodinated rT(3)AM and 3',5'-T2AM at the phenolic rings but in a PTU-insensitive fashion. All thyronamines with tyrosyl ring iodine atoms were 5(3)-deiodinated by Dio3-containing preparations. In functional competition assays, the newly identified thyronamine substrates inhibited an established iodothyronine deiodination reaction. By contrast, thyronamines that had been excluded as deiodinase substrates in LC-MS/MS experiments failed to show any effect in the competition assays, thus verifying the former results. These data support a role for deiodinases in thyronamine biosynthesis and contribute to confining the biosynthetic pathways for 3-T 1 AM and T 0 AM.

Novel Perfluoroalkyl-Diphenylphosphine Compounds. Syntheses and Reaction Mechanisms

A new class of perfluoroalkyl compounds, containing novel Rf−Ph2P bonds (where Rf is the perfluoroalkyl moiety), were synthesized in fairly good yields, and their reaction mechanisms were studied in different solvents. Thus, the reaction of 1-iodo-n-perfluorohexane (1a) in liquid ammonia afforded only the hydro deiodination product (3a), and under irradiation a low yield of diphenyltridecafluorohexylphosphine (2a) was obtained. However, good yields (≥72%) of substitution products were obtained with 1a and 1-iodo-n-perfluorobutane (1c) under irradiation in HMPA and DMPU as solvents. These reactions were completely inhibited with di-tert-butyl nitroxide, a good free radical scavenger. The photostimulated reaction of 1,4-diiodoperfluorobutane (1d) in HMPA afforded the disubstitution product in 55% isolated yield. Low yields of substitution products were obtained with 1a and 1c either in the dark or under irradiation in tetraglyme. It is suggested that the reactions in HMPA and DMPU occur by the SRN1 mechanis...

The selenoenzyme family of deiodinase isozymes controls local thyroid hormone availability.

The human thyroid gland secretes thyroid hormones under the control of the hypothalamus-pituitary-periphery feedback axis already during the last fetal trimester and is the only endogenous source of thyroid hormones after postnatal weaning. The main secretory thyroid hormone product is L-thyroxine (T4) and to a varying extent 3,30,5triiodo-L-thyronine (T3). The latter iodothyronine exerts most of the effects of thyroid hormones on development, differentiation and metabolic pathways via binding to ligand-modulated nuclear or mitochondrial T3-receptors. Direct hormone effects have also been described for T4 and its deiodination products T3 and 3,30,50-triiodothyronine (reverse-T3, rT3) at the plasma membrane and for 3,5-T2 at the mitochondrial level. T4 and its derivatives undergo extensive metabolic reactions in several tissues, organs and cells, the maiority of which are catalyzed by the selenoenzyme family of iodothyronine deiodinases [1]. The three deiodinase isozymes so far identi®ed are expressed in tissue-speci®c and developmentally regulated patterns thus controlling local production and degradation of the thyromimetic hormone T3 and of the other lower iodinated iodothyronines. The deiodinases act as gatekeepers of access of T3 to its intracellular receptors and modulate local availability of T4 and the other iodothyronines for their interaction with cellular targets different from T3-receptors [2]. Type I iodothyronine 50-deiodinase (50DI) preferentially deiodinates T4 and reverse-T3 at the 50-position of the phenolic ring, but also removes iodide from the 5position of the tyrosyl-ring under certain reaction conditions. Type II 50-deiodinase (50DII) deiodinates T4 and with lower af®nity also rT3 speci®cally at the phenolic ring in 50-position. The type III iododthyronine 5-deiodinase (5DIII) removes iodide from the 5-position of the tyrosyl ring and thus catalyzes the inactivation of the thyromimetic activity of T4 and T3.

2,3]-Wittig Rearrangement Initiated by 1,5-Hydrogen Atom Transfer from an o-Iodophenyl Group on the α-Carbon of Allylic Ethers by Reduction with SmI2

Abstract Intramolecular 1,5-hydrogen atom transfer of an aryl radical generated by reduction of an o-iodophenyl group on the allylic position of allyl ethers by SmI2 regioselectively generates α-allyloxy carbanions, which undergo [2,3]-Wittig rearrangement to afford a substituted 4-phenyl-3-buten-1-ol. The effect of HMPA concentration on distribution of the 1,5-hydrogen transfer giving Wittig rearranged products and hydrogen abstraction giving reductive deiodination products is described.

Type 1 deiodinase is stimulated by iodothyronines and involved in thyroid hormone metabolism in human somatomammotroph GX cells.

Local 5'-deiOdination of l-thyroxine (T4) to the active thyroid hormone, 3,3',5-tri-iodothyronine (T3) via two deiodinase isoenzymes (D1 and D2) has an important role for various T3-dependent functions in the anterior pituitary. However, no evidence has been presented yet for thyroid hormone inactivation via the 5-deiodinase (D3) in anterior pituitary models. Using the human somatomammotroph cell line, GX, we analysed effects of T3 and its 5'-deiodination product, 3,5-di-iodothyronine (3,5-T2), on deiodinase activities, measuring release of iodide-125 (125I-) from phenolic-ring- or tyrosyl-ring-labelled substrates respectively. T3 and 3,5-T2 rapidly stimulated D1 activity in GX cells in the presence of serum in the culture medium, whereas D2 activity was not detectable under these conditions. However, when the cells were kept under serum-free conditions, specific activity of D2 reached levels similar to those of D1. With tyrosyl-ring labelled 3, 5-[125I]-,3'-T3 as substrate, a significant release of 125I- was observed in GX cell homogenates. This is comparable to the D1 activity of liver membranes, which preferentially catalyses 5'-deiodination, but to some extent also 5-deiodination, at the tyrosyl ring. D1 activity of human GX cells is increased by T3 and 3,5-T2. Inactivation of T3 in the anterior pituitary might occur by deiodination at the tyrosyl ring via D1, thus terminating the stimulatory thyroid hormone signal in human somatomammotroph cells.

Effect of cold exposure on thyroid hormone metabolism and nuclear binding in rat brain.

In this study we examined whether adult rat brain tissue (cerebral hemispheres) would under cold exposure respond with changes in the local metabolism and nuclear binding of thyroid hormones (T3, T4). Adult, control rats kept at 22 degrees C and cold exposed (4 degrees C, 20 h) rats were injected with trace of 125I-T4 or 125I-T3 returned to their respective environment and sacrificed four hours later. The radioactive hormonal forms were identified and quantified in the cytoplasmic and nuclear fractions. It was found that in cold exposed rats injected with 125I-T4, the total cytoplasmic radioactivity was higher than that of controls. This increase was not associated with 125I-T4 but it reflected an increase (88 %) in its deiodination product 125I-T3 (125I-T3 (T4)). Although total cytoplasmic 125I-T4 did not change, there was a decrease (28%) in its protein free cytoplasmic fraction. 125I-T3 (T4) and 125I-T4 bound to the nuclear fraction were found to decrease by 58 and 46% respectively. Cold exposed animals injected with 125I-T3 also showed an increase in cytoplasmic 125I-T3 (81%) and a decrease in 125I- (40%) whereas 125I-T3 bound to the nuclear fraction decreased by 64%. These results indicate that cold exposure of rats decreases brain local T3 metabolism and nuclear binding while it does not effect local T4 metabolism.

3,5-diiodo-L-thyronine stimulates type 1 5'deiodinase activity in rat anterior pituitaries in vivo and in reaggregate cultures and GH3 cells in vitro.

Local deiodination of L-thyroxine (T4) to the active thyroid hormone T3 via two 5'deiodinase isoenzymes (5'DI and 5'DII) plays an important role for various T3-dependent functions of the anterior pituitary (AP). Recently, it was reported that 3,5-T2, the 5'deiodination product of T3, acts as a specific agonist in the feedback mechanism on TSH secretion at the pituitary level. We now examined the effects of 3,5-T2 on pituitary 5'deiodinase activities in vivo in male, adult rats and in vitro using rat AP reaggregate cultures and the somatomammotroph cell line GH3. 5'DI activity in the AP was transiently increased after a single injection of 3,5-T2. Serum TSH levels declined, and 24 h after 3,5-T2 application, betaTSH steady-state mRNA levels in the APs were markedly lower. In reaggregate cultures of the AP, 3,5-T2 stimulated 5'DI activity 24 h after application, dose-dependently. Compared with 5'DI activities, those of 5'DII were an order of magnitude lower, in vivo as well as in vitro, and were rapidly and transiently decreased by the higher dose of 3,5-T2. GH3 cells responded to 3,5-T2 and T3 by an 1.7-fold stimulation of 5'DI activity. Stimulation of DNA-binding was demonstrated in electrophoretic mobility shift assays for a specific RXR-containing protein complex with a DR+4 thyroid hormone response element of the human type 1 5'DI promoter using nuclear extracts from GH3 cells treated with 3,5-T2. In summary, 3,5-T2 and T3 exert direct thyromimetic effects on 5'DI activity and TSHbeta expression at the pituitary level. 5'DI is regulated by its substrate(s) and/or products and may serve an important function within the modulation of thyroid hormone-dependent gene expression in the AP.

Identification of Thyroid Hormone Conjugates Produced by Isolated Hepatocytes and Excreted in Bile of Rainbow Trout,Oncorhynchus mykiss

We studied thyroid hormone (TH) conjugation in fasted trout by incubating isolated hepatocytes with either [125I]T4or [125I]T3, and by analyzing bile from trout injected with either [125I]T4or [125I]T3. Glucuronide conjugates were identified by hydrolysis with β-glucuronidase and sulfate conjugates by acid solvolysis with ethyl acetate/trifluoroacetic acid (1%). We used Sephadex LH-20 chromatography to concentrate the conjugate fractions from hepatocyte incubates prior to HPLC analysis. Glucuronide conjugates of T4and T3were producedin vitroand glucuronides of T4, T3, and 3,3′-T2were foundin vivo.Sulfation of T4occurredin vitroandin vivo.T3sulfation was not establishedin vitro,but sulfate conjugates of T3and T2were found in bile. Significant proportions of unconjugated T4and T3also occurred in the bile. We conclude that (i) as in other vertebrates, iodothyronines undergo hepatic glucuronidation and are excreted as such in the bile, (ii) T4and T3undergo sulfation, and in contrast to mammals, are excreted in significant amounts in the bile, (iii) 3,3′-T2, a prominent deiodination product of T3, is excreted as both glucuronide and sulfate conjugates, and (iv) the isolated hepatocyte system is appropriate for studying aspects of TH metabolism in trout.

Colon-specific prodrugs of 5-radioiodo-2'-deoxyuridine.

Two glycoside-based prodrugs, 125IUdR-5'-beta-D-glucopyranoside and 125IUdR-5'-beta-D-galactopyranoside, were synthesized. This selection was dictated by the abundance of appropriate enzymes in the GI tract of mice and similar levels of beta-D-glycosidases in human and rodent large intestine. Studies to establish the ability of colonic microflora to release 125IUdR were conducted in vitro and in Swiss Webster mice. Both prodrugs released 125IUdR in the presence of the corresponding enzymes or the GI content homogenates in vitro, and in vivo. Luminal enzymes in the proximal and distal small intestine in mice degraded less than 10% of each prodrug whereas enzymes from the colonic/caecal lumen of mice released nearly 100% of 125IUdR. 125IUdR freed by bacterial glycosidases was stable in the GI content. No significant amounts of other metabolites or deiodination products were observed. Total radioactivity recovered as by-products was less than 10%. The efflux of prodrugs from the GI tract after oral administration in mice was slow and limited. Unlike 125IUdR, prodrugs were not dehalogenated in vivo as indicated by biodistribution and imaging studies.

Studies on the rapid stimulation of mitochondrial respiration by thyroid hormones.

Injection of L-3,5-diiodothyronine (T2) into rats made hypothyroid by 6-n-propyl-2-thiouracil (PTU) increased the respiration rates of subsequently isolated liver mitochondria; this stimulation of respiration by T2 occurred in the presence of cycloheximide and is therefore independent of protein synthesis on cytoplasmic ribosomes. Injection of T3 into PTU-treated rats had a lesser effect than T2 on the respiration rates of subsequently isolated mitochondria; as PTU is an inhibitor of 5'-iodothyronine deiodinases, which convert T3 into T2 in vivo, the rapid stimulation of mitochondrial respiration by T3, which has been shown in a range of systems, may not be due directly to T3 itself, but may be mediated by its deiodination product T2. Injection of T2, or T3, into hypothyroid or euthyroid rats had no effect on the percentage activity of mitochondrial pyruvate hydrogenase assayed 30 min later. The amount of active pyruvate dehydrogenase is regulated by changes in mitochondrial calcium concentration and matrix ATP/ADP ratio; therefore these parameters are not persistently affected by treatment with T3 or T2. In addition, the total amount of pyruvate dehydrogenase present was the same in euthyroid and hypothyroid rats, indicating that the expression of this enzyme is not stringently controlled by thyroid hormone status.

HPLC analysis of in vitro hepatic deiodination products of thyroid hormones in the rainbow trout, Oncorhynchus mykiss

Reverse-phase HPLC employing five different solvent systems was used to determine the 125I-labeled products formed in rainbow trout by in vitro incubation at 12° of the hepatic microsome fraction with l-thyroxine (T 4) labeled with 125I in the outer phenyl ring. Trout were starved for 2 weeks or fed a 2% ration. The only labeled products identified during incubation for 7.5–70 min over a T 4 substrate range of 0.03–0.5 n M were 125I − and 3,5,[ 125I]3′-triiodo- l-thyronine (T 3). These products in combination with the parent [ 125I]T 4 accounted for over 96% of the total chromatographic radioactivity. Neither 3,[ 125I]3′-diiodo- l-thyronine nor 3,[ 125I]3′,5′-T 3 (reverse T 3) was detected, suggesting negligible inner-ring deiodination of T 4 or T 3. Essentially equal production of 125I − and [ 125I]T 3 validated the use of 125I − production as a measure of [ 125I]T 3 generation in assays for hepatic 5′-monodeiodinase activity. However, in some experiments the 125I − level slightly exceeded the [ 125I]T 3 level, indicating that outer-ring deiodination of T 3 may occur to a limited degree. In conclusion, the present data for liver support earlier observations from in vivo studies in showing that for trout at 12° deiodination pathways are geared primarily toward T 4 outer-ring monodeiodination to produce T 3 with undetectable inner-ring deiodination of T 3 or T 4 and limited outer-ring deiodination of T 3.

Thyroxine monodeiodination in normal human kidney tissue in vitro.

The present study deals with thyroxine monodeiodination in normal human kidney. To allow for comparison with previous reports, the present methods are similar to those used by others in rat tissue studies. The microsomal cell fraction of normal human kidney tissue was obtained by differential ultracentrifugation. The microsomes were incubated under various conditions and the deiodination products assayed with radioimmunoassay. A type I 5'-monodeiodinase was demonstrated, pH optimum around 6.5. Competitive inhibition was observed of T3 generation from T4 by rT3 with a Km of 3.0 microM and a Ki of 4 microM. Vmax was 26.1 pmol/min/mg protein. Likewise rT3 was generated from added T4, but it was rapidly degraded, while T3 was relatively stable as is the case in rat tissue preparations. Propylthiouracil inhibited 5'-deiodination in a dose dependent fashion with complete abolishment of deiodination at propylthiouracil concentration of 10(-4) M. Ipodate inhibited the reaction with complete inhibition at 10(-2) M. The data demonstrate that a human kidney particulate cell-fraction contained considerable amounts of T4 deiodinases, very similar to the type I deiodinase of various rat tissue, although the handling of rT3 and the inhibitory action of this iodothyronine on T4 to T3 conversion seem to be slightly different in the two species.

Thyroxine metabolism in cultured cells derived from fetal rat lung.

Summary: The role of thyroxine (T4) in fetal lung development has been demonstrated previously. Because triiodothyronine (T3), not T4, is thought to be the active hormone, and because T3 nuclear receptors appear to be present in fetal lung, the following studies were performed to examine peripheral metabolism of [125I]T4 by organotypic and mixed monolayer cultures of fetal lung cells. The cells were derived from fetal rat lungs obtained at 16 and 19 days of gestation.Short term (2 h) incubations of homogenized organotypic cultures and mixed monolayer cultures with [125I]T4 showed minimal amounts of [125]T3 generation expressed as % added T4 per mg protein (0.43 ± 0.32 and 0.56 ± 0.28, respectively). In addition, there appeared to be “excess iodide” (2.21) and substantial generation of rT3 (i.e., 2.67 ± 1.1 and 1.87 ± 1.0, respectively).In a subsequent group of experiments, deiodination of [125I]T4 was allowed to proceed by incubating intact organotypic cultures with [125IT4 for longer periods (36 h). Subsequent assessment and comparison of deiodination products in the subcellular fractions showed that the highest % of [125I]T3 (relative to total thyronines) was present in the fraction enriched with nuclei (7.7 ± 2.3). This was significantly different from the [125I]T3 in homogenized whole cells (4.54 ± 1.5), and markedly different from the medium which contained the lowest % of [125I]T3 (0.18 ± 0.2). Further confirmation was obtained to show that this high concentration of [125I]T3 in the nuclear fraction had in fact been generated de novo and did not merely result from redistribution of labeled hormones among the cellular compartments. Incubation of organotypic cultures with two different isotopically-labeled hormones ([131I]T3 and [125I]T4) permitted calculation of nuclear to medium ratios for [131I]T3 and [125I]T3; the ratio of these provided an index of intracellular conversion of [125I]T4 to [125I]T3. The mean of five such experiments demonstrated that of the total [125I]T3 attached to the nuclear fraction at the end of the incubation, 32% was newly generated and 68% was derived by influx from the medium. These results indicate that organotypic cultures derived from day 19 fetal rat lung are capable of deiodinating T4 and that both rT3 and T3 are generated. The generated T3 appears to be retained by the lung cells and is bound to the nuclear fraction. On the other hand, rT3 is distributed more evenly throughout the cell and in the medium and presumably is not bound.

Conversion of T3 and rT3 to 3,3'-T2: pH dependency.

In the extrathyroidal deiodination of T4 the importance of T3 and rT3 for the peripheral action of thyroid hormones is well documented. With the development of a specific radioimmunoassay for 3,3'-T2, a deiodination product of both T3 and rT3, we were able to characterize these subsequent enzymatic reactions as well as the degradation of 3,3'-T2 in rat liver homogenate. It was found that the reaction T3 leads to 3,3'-T2 is slow compared to the conversion of T4 to T3. The pH activity profile shows a peak at 8.4. The reaction rT3 leads to 3,3'-T2 is very fast, with an apparent KM of 4 x 10(-7) M. The reaction velocity is significantly higher in acid than in alkaline pH. The deiodination of 3,3'-T2 is also faster in the acid than in the alkaline range. It is concluded that the outer ring of T4 is more readily deiodinated in acid and the inner ring in alkaline media, and that 3,3'-T2 is mainly degraded to 3-T1. In the acid pH range T3 may accumulate and in the alkaline range rT3 by the pH characteristics of these ractions. Therefore small shifts in pH can enhance the potent inhibitory action of rT3 on the 5'-deiodination of T4 adding another mechanism to the peripheral regulation of thyroid hormone activity.

Thyroid Hormonelike Actions of 3,3′,5′-l-Triiodothyronine and 3,3′-Diiodothyronine

l-Thyroxine is converted to 3,5,3'-l-triiodothyronine (T(3)) as well as to 3,3',5'-l-triiodothyronine (reverse T(3)). One product of further deiodination is 3,3'-diiodothyronine (3,3'T(2)). The serum levels of reverse T(3) and 3,3'T(2) change considerably in various physiological and disease states. We previously found that reverse T(3) and 3,3'T(2) bind to the solubilized hepatic nuclear "receptors" for thyroid hormones. This led us to study binding and actions of these metabolites in cultured rat pituitary cells in which glucose consumption and growth hormone production are regulated by T(3) and l-thyroxine. Reverse T(3) and 3,3'T(2) stimulated growth hormone production and glucose consumption and inhibited nuclear binding of radioactive T(3). Either metabolite produced maximal effects that equaled those of T(3), and neither inhibited the T(3) response. Further, additive effects were observed when reverse T(3) was combined with submaximal concentrations of T(3). In serum-free and serum-containing media, concentrations of 3,3'T(2) 50- to 70- and 10- to 100-fold greater, respectively, than those of T(3) were required for equivalent stimulations and for inhibition of nuclear binding by T(3). The relative activity differences under the two conditions can be attributed to weaker serum protein binding of 3,3'T(2) than T(3). With cells in serum-free media, reverse T(3) was a less avid competitor than 3,3'T(2) for T(3) binding by the nuclear receptors, and was less potent than 3,3'T(2) (0.001 the potency of T(3)) in inducing growth hormone production or glucose oxidation. In incubations with serum-containing media, reverse T(3) was an ineffective competitor for T(3) binding, and had only 0.1 the inducing potency of 3,3'T(2) (0.001 the potency of T(3)). The weaker activity of reverse T(3) relative to 3,3'T(2) in serum-containing media could be explained by stronger serum binding of reverse T(3) than 3,3'T(2). In addition, after long-term incubation of cells with radioactive reverse T(3), much of the cell-associated radioactivity was recovered as 3,3'T(2). These studies suggest that reverse T(3) and 3,3'T(2) can stimulate thyroid hormone-regulated functions as weak agonists by acting via the same receptors that mediate T(3) actions. Moreover, some of the effects of reverse T(3) may be due to 3,3'T(2) produced by deiodination of reverse T(3).

Photochemical reactions of N-phenylacetyl and N-(2-iodophenylacetyl) derivatives of 1,2,3,3a,4,5-hexahydroindol-6-one

Irradiation of 1,2,3,3a,4,5-hexahydro-N-(2-iodo-4,5-dimethoxyphenylacetyl)indol-6-one (2) gave 3,3a,4,5-tetrahydro-10,11-dimethoxyindolo[7,1-ab][3]benzazepine-1,7(2H,8H)-dione (3) and the deiodination product (4). The latter (4) then underwent a photo-Fries rearrangement to 1,2,3,3a,4,5-hexahydro-7-(3,4-dimethoxy-phenylacetyl)indol-6-one (5), which, upon further irradiation, gave 3,3′,4,4′-tetramethoxybibenzyl (13), 2,3,3a,4,-5,6-hexahydro-6-oxo-1H-indole-7-carbaldehyde (12), and -7-carboxylic acid (14). The production of compounds (12)–(14) is accounted for by a mechanism involving a type I cleavage and subsequent keten formation. The presence of a keten intermediate was demonstrated by nucleophilic addition of water, an alcohol, or an amine.

Serum Triiodothyronine Measurements by the Sterling Method : A Critical Evaluation

The Sterling method for measurement of triiodothyronine (T3) in serum has been modified to improve separation of thyroxine (T4) and T3 by prolonging the time of chromatography. By altering the elution procedure, less unwanted competitive binding material was eluted from the paper strips, resulting in a lower “blank effect” in the zero tube of the binding assay. A more sensitive binding assay was found by using a granular resin (Rexyn 202, Cl-SO4, Fisher) and lowering the serum concentration in the binding solution. Possible sources of error were evaluated and their magnitude was calculated. Deiodination of T4 to T3 occurs during the assay procedure, but it is small in the range of normally occurring T4 levels (about 0.5% of T4). Contamination of the T3 peak with T4 (or other deiodination products) can be avoided if proper care is taken during chromatography. When the immunoassay procedures are not readily available, this assay can give valuable information to the physician confronted with a difficult diagnostic problem in a patient suspected to have disease of the thyroid gland.