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Endocrinology Vol. 143, No. 5 1789-1800
Copyright © 2002 by The Endocrine Society

TRH-TSH-THYROID

Concentrations of Seven Iodothyronine Metabolites in Brain Regions and the Liver of the Adult Rat

Graziano Pinna, Oliver Brödel, Theo Visser, Andreas Jeitner, Hannah Grau, Murat Eravci, Harald Meinhold and Andreas Baumgartner

Departments of Radiology and Nuclear Medicine (Radiochemistry) (G.P., O.B., A.J., H.G., M.E., H.M., A.B.), Universitätsklinikum Benjamin-Franklin, Free University of Berlin, Berlin 12200 Germany; and Department of Internal Medicine III, Erasmus University Medical School (T.V.), 3062 PA Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Andreas Baumgartner, M.D., Department of Radiology and Nuclear Medicine (Radiochemistry), Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: . Abaum{at}cipmail.ukbf.fu-berlin.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Hormone determination
 Results
 Discussion
 References
 
The concentrations of the iodothyronine metabolites T4, T3, 3,5-diiodothyronine (3,5-T2), 3,3'-diiodothyronine (3,3'-T2), reverse T3 (rT3), 3,3'-T2 sulfate (3,3'T2S), and T3 sulfate (T3S) were measured in 12 regions of the brain, the pituitary gland, and liver in adult male rats. Quantification of iodothyronine was performed by RIA following a newly developed method of purification and separation by HPLC. 3,5-T2, 3,3'-T2, rT3 and T2S were detectable in the low femtomolar range (20–200 fmol/g) in most areas of the rat brain. T3S was detectable only in the hypothalamus. The concentrations of T3 and T4 were approximately 20- to 60-fold higher, ranging between 1 and 6 pmol/g. There was a significant negative correlation between the activities of inner-ring deiodinase and T3 concentrations across brain areas. In the liver, 3,5-T2, rT3, and T3S were measurable in the low femtomolar range, whereas 3,3'-T2 and 3,3'T2S were not detectable. 3,5-T2 and 3,3'-T2 were not detectable in mitochondrial fractions of the brain regions. Tissue concentrations of 3,5-T2 exhibited a circadian variation closely parallel to those of T3 in the brain regions and liver. T3 was not a substrate for outer-ring deiodination under different experimental conditions; thus, it remains unclear which substrate(s) and enzyme(s) are involved in the production of 3,5-T2. These results indicate that five iodothyronine metabolites other than T3 and T4 are detectable in the low femtomolar range in the rat brain and/or liver. The physiological implications of this finding are discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Hormone determination
 Results
 Discussion
 References
 
THE CLASSICAL EFFECTS of thyroid hormones are exerted by binding of T3 to its specific nuclear receptors, thereby influencing the expression of target genes (for a review see Ref. 1). Such effects have now also been demonstrated in the adult vertebrate brain (e.g. Ref. 2). Other iodothyronine metabolites may, however, also affect the brain by acting via nonnuclear mechanisms. T4, for example, promotes polymerization of actin (e.g. Ref. 3). Reverse T3 (rT3) has been found to be five times as potent as T4 in inhibiting type II outer-ring deiodinase (D2) activity in rat cortex (4). Physiological effects of other iodothyronine metabolites have been described in various tissues but have not yet been investigated in the brain. 3,5-diiodothyronine (3,5-T2), for instance, has been reported to enhance the activity of hepatic mitochondrial cytochrome oxidase (5) and oxygen consumption (6), suppress TSH in rat pituitary glands (7, 8), stimulate type I deiodinase (D1) activity in rat anterior pituitary (9), and also enhance liver glucose 6-phosphate dehydrogenase activity (10). A calorigenic effect of 3,3'-diiodothyronine (3,3'-T2) has also been reported by Goglia’s group (e.g. Ref. 11).

However, to our knowledge, the tissue concentrations of rT3, 3,5-T2, and 3,3'-T2 have not yet been directly measured in rat tissues such as the liver or brain.

Furthermore, it is as yet unclear whether sulfated iodothyronines occur in the brain under physiological conditions, although two reports indirectly indicate an existence of T3 sulfate (T3S) and 3,3'-T2 sulfate (3,3'-T2S) in the CNS. Hurd et al. (12) measured the sulfation of T3 in cytosolic fractions in vitro and found high sulfotransferase activity in rat liver, brain, and kidney. Esfandiari et al. (13) reported that [125I]T3 is metabolized to 3,3'-T2S, but not to T3S in cultured astrocytes. Moreover, a substantial body of data provides evidence that sulfation of both T3 and 3,3'-T2 occurs in rat hepatocytes (e.g. Refs. 14, 15 ; for a review see Ref. 16). However, tissue levels of T3S and 3,3'-T2S have also never been directly measured in rat brain or liver.

One purpose of the present study was therefore for the first time to determine whether the hormones 3,5-T2, 3,3'-T2, rT3, T2S, and T3S are detectable in various regions of the brain and the liver of the rat. For comparison, we also quantified T3 and T4 in the same tissues. This had been done previously by Morreale’s group in the cortex, cerebellum, and kidney (e.g. Ref. 17) and by our own group in 11 regions of the brain and the liver (e.g. Ref. 18). However, in our own psychopharmacological studies even the control rats had usually received some kind of treatment, e.g. saline ip or orally. Such injections may, however, affect both tissue levels of thyroid hormones and deiodinase activities (19). We therefore again measured T3 and T4 concentrations as well as the activities of the three deiodinase isoenzymes in various brain regions and the liver in untreated control rats.

Morreale de Escobar (20) developed a reliable method for measuring T3 and T4 in rat brain tissue. This method comprises a relatively complicated extraction procedure for the removal of lipids and further purification of the extracts by column chromatography. This method does not separate the iodothyronines and is therefore not suitable for measuring small amounts of several different iodothyronines from the same brain regions. To simplify the methodological procedure and to permit the measurement of several different iodothyronines from the same brain areas, we developed a method for purifying and separating the different iodothyronines by HPLC.

In the course of the study, we detected small but clearly measurable amounts of 3,5-T2 and 3,3'-T2 in some brain areas. We thus further investigated whether the concentrations of these hormones were particularly enhanced in mitochondrial fractions of the respective brain regions, as suggested by previous reports (e.g. Ref. 21). As nothing is known about the metabolic pathways leading to the production of 3,5-T2, we investigated whether T3 is a substrate for outer-ring deiodination in regions of the rat brain under differing experimental conditions.

Moreover, we previously reported diurnal variations in tissue concentrations of T3 in regions of the rat brain and liver (22). We therefore investigated whether the concentrations of 3,5-T2, which is claimed to have physiological functions (see above), also exhibit circadian variations in different brain areas and the liver.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Hormone determination
 Results
 Discussion
 References
 
Materials
T4, T3, rT3, 3,3'-T2, and 3,5-T2 of the highest available purity were obtained from Henning GmbH (Berlin, Germany). 5'-125I-T4, 3'-125I-T3, and [125I]-3,3'-T2 were prepared for iodothyronine deiodinase assays and RIAs by radioiodination of T3, 3,5-T2, and 3-T1, respectively, as previously described (23, 24, 25). The tracers with specific radioactivities of 50–75 megabecquerels (MBq)/nmol were repurified immediately before use with disposable Sep-Pak C18 cartridges (Waters Associates, Milford, MA) yielding a purity >99% with 125I- as the only contaminant. Inner-ring labeled [5-125I]-T4, [5-125I]-T3, and 3-Br-[5-125I]-T1 (specific radioactivity 1.0–1.5 MBq/nmol) were purchased from R. Thoma (Formula GmbH, Berlin). Dithiothreitol (DTT) was purchased from Boehringer GmbH (Mannheim, Germany). 6-n-propyl-2-thiouracil (PTU); iopanoic acid (IOP); aurothioglucose; ß-nicotinamide adenine dinucleotide (NAD); NAD phosphate (NADP); NAD phosphate, reduced form (NADPH); NAD, reduced form (NADH); and flavin adenine dinucleotide (FAD), were purchased from Sigma (Munich, Germany). The HPLC column (Eurosphere 100 C18) was bought from Knauer (Berlin, Germany). All other chemicals were of reagent grade.

Animals
All of the animal experiments described in this study were evaluated and approved by the Animal Protection Committee of the Berlin Senate. Adult male euthyroid Sprague Dawley rats weighing 250–300 g were employed throughout. They were housed in individual cages on a 12-h light, 12-h dark schedule (lights on at 0600 h) and had access to food and water ad libitum. All rats were decapitated without anesthesia. Their brains were dissected according to Glowinski and Iversen (26). The pituitaries and livers were also removed and all tissues stored immediately at -70 C. Blood was drawn from the decapitation wound and centrifuged, and the serum was stored at -20 C. Unless elsewhere stated, all rats were decapitated between 1100 h and 1300 h. Following an adjustment period of at least 1 wk in the new environment, the following studies were performed.

Group 1: thyroid hormone concentrations in homogenates of different brain regions, the pituitary gland and liver
Two groups each comprising 8 rats served for the quantification of iodothyronines in homogenates of 12 areas of the brain (olfactory bulb, frontal cortex, parieto-occipital cortex, hippocampus, amygdala, septum, limbic forebrain, striatum, midbrain, hypothalamus, cerebellum, and medulla), pituitary gland and liver.

Group 2: mitochondrial concentrations of T2
One group of 8 rats was used to determine T2 concentrations in mitochondria. As neither 3,5-T2 nor 3,3'-T2 was detectable in four pools, each containing two mitochondrial fractions from a single brain region, an additional group of 36 rats was killed to further investigate the concentrations of 3,5-T2 and 3,3'-T2 in three pools each containing 12 mitochondrial fractions each for a single hormone determination.

Group 3: deiodinase activity in homogenates of different brain regions, the pituitary gland and liver of the rat; investigation of outer-ring deiodination from T3 to 3,5-T2
Eight rats were decapitated to measure D1, D2, and D3 in the different brain areas reported above, and the pituitary glands and livers. In addition, the outer-ring deiodination of T3 to 3,5-T2 was investigated under different experimental conditions in another group of six rats.

Group 4: circadian variations of iodothyronines
One group of 36 male euthyroid rats were kept on a 12-h light, 12-h dark schedule (lights on at 0600 h), under standard laboratory conditions and were decapitated at 4-h intervals (six animals each) over a 24-h period. During the dark phase of the 24-h cycle, the animals were killed under a dim red light (3–5 lux).

Subcellular fractionation
The method used to isolate the mitochondria was a modification of the methods described by Dodd et al. (27). All procedures were carried out at 4 C room temperature. Frozen tissue was placed in ice-cold 0.32 M sucrose at a final dilution of 1:10 (wt/vol). The tissue was homogenized mechanically with a motor-driven glass-Teflon homogenizer (Braun Melsungen AG, Melsungen, Germany) and cooled in an ice-water mixture throughout. The clearance between tube and pestle was 0.2 mm, the motor speed 750 rpm, and 20 strokes of the pestle were required for full homogenization of the tissue sample.

The centrifugation steps were carried out in an L8–55 ultracentrifuge (Beckman Coulter Instruments) using a swinging bucket rotor SW 41 Ti (Beckman Coulter Instruments). The tissue homogenate was centrifuged at 2,600 rpm for 15 min to obtain the low-speed supernatant (S1) and the crude nuclear pellet. The supernatant S1 was diluted with 0.32 M sucrose to yield a final volume of 9 ml, layered directly onto 4 ml of 1.2 M sucrose, and centrifuged at 200,000 x g for 30 min. After subcellular fractionation, the isolated mitochondria were washed for 1 min in a vortex mixer with a solution of 0.32 M sucrose containing 25 µM IOP and then centrifuged for 15 min at 14,000 rpm and 4 C. The supernatants were discarded and the pellets, which consisted of mitochondria, were stored at -85 C until use. The tissue of a single rat was used for each mitochondria preparation. The purity of the mitochondrial fractions was determined by electron microscopy and by measuring cytochrome oxidase activity (using the method described by Hevner et al., Ref. 28), both in an aliquot of the homogenate and in the mitochondrial fraction. Electron microscopy showed approximately 80% purity of the fractions and the determinations of cytochrome oxidase activity revealed a 5-fold higher concentration in the mitochondrial fraction than in the homogenate (data not shown).


   Hormone determination
Top
 Abstract
 Introduction
 Materials and Methods
 Hormone determination
Results
 Discussion
 References
 
RIA buffers
Phosphate buffer (0.04 M, pH 8.0) containing 243 mg/liter merthiolate and 2 g/liter BSA (for the T3, rT3, 3,5-T2, and 3,3'-T2 RIAs) and 0.5 g/liter BSA (for the T4 RIA) served as experimental buffers. The iodoamino acids were dissolved in 0.1 M sodium hydroxide and diluted to the final assay concentrations using this buffer. The [125I]T4, [125I]T3, [125I]rT3, 3-Br-[5-125I]T1, and [125I]3,3'-T2 tracers were each dissolved in the respective experimental buffer containing 100 mg/liter L-cysteine. The stop solution consisting of the phosphate buffer, 243 mg/liter merthiolate, 30% (wt/vol) polyethylene glycol, and 1.3 mg/ml bovine {gamma}-globulin was pipetted (3 ml/tube) to precipitate the antibody-bound radioactivity in the T4, rT3, T3, 3,3'-T2, and 3,5-T2 RIAs. The RIA buffer for T3S and 3,3'-T2S consisted of 0.06 M barbital (pH 8.6), 0.15 M NaCl, and 0.01% (wt/vol) BSA.

Synthesis of rT3 tracer
Labeled rT3 of maximum specific radioactivity was obtained by radioiodination of 3,3'-T2 with chloramine-T (29). 3,3'-T2 was dissolved in 0.01 mol/liter sodium hydroxide and further diluted in 0.1 mol/liter phosphate buffer (pH 7.0). Chloramine-T (Merck, Darmstadt, Germany) and sodium disulfite were also diluted in the phosphate buffer. For labeling, 10 µl of the following reagents were pipetted into a microflex vial containing 40 µl 0.5 mol/liter phosphate buffer (pH 7.5): 37 Mbq (1 mCi) 125iodine, 2 nmol 3,3'-T2, 0.2 µmol chloramine-T. After an incubation period of 30 sec, 1.5 µmol sodium disulfite were added. The reaction mixture was injected onto the HPLC column. HPLC separation of [125I]rT3 was carried out using a 5-µm Eurospher 100-C18, 4 x 250 mm column (Knauer GmbH). The column was equilibrated with a gradient of 55% methanol and 45% 0.02 M ammonium acetate buffer, pH 4.0, at a flow rate of 1 ml/min. [125I]rT3 eluted from the column after approximately 12 min. The fraction eluting from the HPLC column, which corresponded to the [125I]rT3 peak (about 75% of the total radioactivity used for the labeling) was collected and diluted with 100% methanol containing 0.25% concentrated ammonia. This solution was stored at -20 C. Specific radioactivity ranged from 1040–1550 Ci/mmol.

Preparation of rT3-binding antibody
Antiserum to rT3 was produced in three rabbits immunized by serial injections of rT3 conjugated to BSA in complete Freund’s adjuvant as described previously (30). All immunized rabbits produced antisera suitable for RIA of rT3. The antiserum used in the experiments was obtained after three injections of the immunogen, 8 wk after starting immunization. The final dilution was 1:150,000, which bound about 40% of tracer in an incubation volume of 250 µl.

RIA procedure for rT3 in tissue samples
The RIA of tissue rT3 was carried out in 10 x 55 mm plastic tubes with the addition of various reagents as follows: 1) 50 µl rT3 standard at concentrations ranging from 1.0 to 75 fmol/tube; 2) 100 µl antibody against rT3; 3) 100 µl tracer solution containing about 4000 cpm [125I]rT3; 4) 50 µl of the tissue sample dissolved in experimental buffer; and 5) experimental buffer to yield a final standard solution volume of 250 µl/tube.

After 24-h incubation at room temperature, the antibody-bound iodothyronine was precipitated by adding 3 ml polyethylene glycol stop solution to each tube, then centrifuged at 3,000 rpm and 4 C for 45 min. The supernatant was decanted and discarded. The tubes were inverted for 10 min on blotting paper to absorb the remaining liquid. Finally, the tubes were placed in a {gamma}-counter, and the precipitated, bound radioactivity was counted.

Synthesis of 3,3'-T2 sulfate and T3 sulfate tracers
3,3'-[125I]T2 sulfate and [125I]T3 sulfate were synthesized as previously reported with some modifications (31). In brief, solutions of labeled 3,3'-T2 or T3 were evaporated to dryness under a stream of N2 at about 40 C. Two hundred microliters chlorosulfonic acid (ClSO3H) were added to 800 µl dimethylformamide in cooled tubes and immediately vortex mixed. Two hundred microliters of the dimethylformamide/ClSO3H solution were pipetted into the tube containing the dry labeled iodothyronine. After vortex mixing, the solution was incubated for 2 h at 37 C and subsequently at room temperature overnight. The reaction was then stopped by placing the tubes on ice and adding 800 µl ice-cold H2O.

The solution was then filtered using minisart SRP 15 filters (0.2-µm PTFE-membrane) and injected onto an HPLC column. HPLC separation of [125I]T3 sulfate and 3,3'-[125I]T2 sulfate was carried out using a 5-µm Eurospher 100-C18, 4 x 250 mm column (Knauer GmbH). The column was equilibrated with a gradient of 55% methanol and 45% 0.02 M ammonium acetate buffer, pH 4.0 at a flow rate of 1 ml/min. 3,3'[125I]T2 sulfate and [125I]T3 sulfate eluted from the column after 9 and 17 min, respectively. The fraction eluting from the HPLC column which corresponded to the 3,3'[125I]T2 sulfate and [125I]T3 sulfate peak was collected and stored at 4 C.

Determination of iodothyronines in tissue samples
The tissue concentrations of T4, rT3, T3, 3,3-T2, 3,5-T2, T3 sulfate and 3,3'-T2 sulfate were determined after extraction, HPLC purification and separation of the iodothyronines from the tissue samples using a newly developed method. In brief, various areas of the brain, pituitary and liver were homogenized with 3 ml 100% methanol. The mitochondrial fractions were resuspended in 1.2 ml 100% methanol. Radiolabeled iodothyronines (T4, rT3, T3, 3,3'-T2, 3,5-T2, T3 sulfate, and 3,3'-T2 sulfate ~350 cpm/100 µl) were added to each sample (100 µl to each tube) to measure their recovery and identify the iodothyronine of interest after extraction and HPLC separation. The samples were then placed in a vortex mixer for 1 min and centrifuged for 30 min at 3,000 rpm and 4 C. The supernatants were collected, filtered once (in smaller brain regions) or twice (in larger brain areas) through a 0.2-µm filter, dried by vacuum centrifugation, and resuspended in a solution of 38% methanol and 62% ammonium acetate buffer (0.02 M, pH 4) before injection onto the HPLC column.

The tissue samples were run together with ~10% of their number, of which were handled in exactly the same manner as the tissue samples. The pellets were stored for protein measurement as previously described (32).

As 3,5-T2 and T3 sulfate proved to be extremely difficult to separate by HPLC, two different HPLC runs were performed. For this purpose, two different groups of rats were killed (see above) and extracted separately. In the first extraction radioactively labeled T2S, T3S and rT3 were added and these hormones were separated by HPLC. In the second run, the same was done for 3,5-T2, 3,3'-T2, T3 and T4. For both runs, a Eurosphere 100 C18 column from Knauer (Berlin, Germany) was used. The eluent consisted of a gradient of methanol and ammonium acetate buffer (0.02 M, pH 4), with a linear increase from 38% methanol at min 0 to 42% methanol at minute 30 at a flow rate of 1 ml/min. Between min 31 and 38 the column was washed with 100% methanol at a flow rate of 1.5 ml/min. Between min 39 and 45, the equilibrium of 38% methanol was reestablished, again at the flow rate of 1.5 ml/min. Under these conditions, each hormone peaked at between approximately 2 and 4 fractions of 1 ml each and the individual hormones were clearly separable from each other. Our attempts to use an eluent of acetonitrile and water (including a solution of 1% acetic acid with a pH of 2.8) resulted in almost complete deiodination of 3,3'T2S and T3S. However, such complete deiodination was only seen when tissue samples were processed, not when preliminary experiments were done with the eluent containing only the radioactivity. After HPLC purification and separation, the respective fractions were collected in glass tubes according to their known radioactivity peaks, evaporated to dryness by vacuum centrifugation and taken up in the experimental buffer before RIA. The samples from the brain, pituitary, and liver were all subjected to the same HPLC process.

T4, T3, 3,3'-T2, and 3,5-T2 RIAs were performed as reported previously (23, 24, 25). The RIAs for T3S and 3,3'-T2S were performed as follows. 123I-labeled and unlabeled T3S and 3,3'-T2S were prepared as previously described (31). T3S antiserum Wu-021 (33) and 3,3'-T2S antiserum Wu-0215 (34) were kindly provided by Dr. Sing-Yung Wu (Department of Nuclear Medicine and Medicine Services, Veterans’ Affairs Medical Center, Long Beach, CA). They were used at final dilutions of 1:60,000 and 1:20,000, respectively.

Samples were reconstituted in 250 µl RIA buffer and 100-µl aliquots were assayed in duplicate. Assay mixtures contained 1) 20,000 cpm labeled ligand; 2) 0.5–500 fmol unlabeled ligand (standard) or reconstituted sample; and 3) diluted antiserum in a final volume of 500 µl RIA buffer. After incubation for 3 d at 4 C, antibody-bound radioactivity was isolated using Sac-Cel second antibody-coated cellulose suspension (Immunodiagnostic Systems Ltd., Boldon, UK). The ED90, ED50, and ED10 values in the T3S RIA were 1.5, 16, and 173 fmol and those in the 3,3'-T2S RIA 1.7, 15, and 130 fmol.

Cross-reactivities in the T3S RIA were T4S 10.9%, 3,3'-T2S 2.8%, rT3S 0.7%, T3 <0.01%; in the 3,3'T2S RIA: T3S 25.6%, rT3S 2.25%, T4S 0.01%, 3,3'-T2S 0.04%. However, because T3S and 3,3'-T2S were determined in HPLC-purified samples, these cross-reactivities are not relevant.

All samples of one experiment were processed in duplicate and assayed together within the same run. The results were corrected on the basis of the individual recovery data. For this purpose, the same amount of radioactivity as was added to the tissue samples before extraction was placed in three tubes per hormone, kept in the refrigerator at -20 C, and measured in the {gamma} counter together with the residual radioactivity present in all tissue samples after the HPLC run and immediately before the RIA.

The stabilities of all hormones under investigation were tested in separate experiments during the HPLC separation and drying procedure. A known amount of radioactivity from each hormone (e.g. 500 cpm) was processed through the HPLC column, dried, and again resuspended and processed through the HPLC column to investigate whether deiodination had occurred during all these procedures. Furthermore, a known amount of each hormone (e.g. 2.5 or 5 pg T3) was processed through the HPLC column and quantified by RIA to establish whether a relevant amount of hormone is lost during the HPLC procedure.

Iodothyronine deiodinase assays
The deiodinase activities were measured as previously described (e.g. Ref. 18). The tissue samples were homogenized individually on ice in 5–6 vol 0.25 M sucrose, 10 mM HEPES (pH 7.0) containing 10 mM DTT, immediately frozen in a dry ice/acetone bath, and stored at -80 C until assay. The measurement of D1, D2, and D3 was based on the release of radioiodide from the 125I-labeled substrates.

D1 and D2 assay.
The activity of D1 was determined by measuring the release of radioiodide from 100,000 cpm (~2.5 kBq) (5'-125I)rT3 at 5 nM rT3, 20 mM DTT, in the presence (for D2) and absence (D1 + D2) of 1 mM PTU. D2 activity was determined using (5'-125I)T4 as substrate in the presence of 6 nM T4, 30 mM DTT, 1 mM PTU, and 1 µM T3, to inhibit the inner ring deiodination of T4 in the tissues containing significant D3 activity.

The measurement was conducted after 45–90 min (usually 60 min) incubation at 37 C with 50–100 µg of protein from the crude homogenate in 100 µl 0.1 M potassium phosphate buffer (pH 7.0), 1 mM EDTA. The reaction was started by the addition of the tissue homogenate and stopped by adding 50 µl ice-cold 5% BSA and 10 mM PTU, followed by 400 µl 10% ice-cold trichloroacetic acid. After centrifugation at 4, 000 x g for 30 min, the supernatant containing the 125I- was further purified by cation exchange chromatography. The iodide was then eluted with two 1-ml aliquots of 10% acetic acid and the radioactivity was counted in a {gamma}-counter.

D3 assay.
For determination of D3 (inner-ring deiodinase) 20–70 µg protein were incubated in a final volume of 100 µl 0.1 M potassium phosphate buffer (pH 7.4), 1 mM EDTA with approximately 1.2 kBq (~50,000 cpm) inner-ring labeled [5-125I]T3, at 50 nM T3, 20 nM DTT, and 1 mM PTU for 60 min at 37 C. Radioiodide release was measured as described above.

Outer-ring deiodination of T3
The outer-ring deiodination of T3 was investigated in homogenates of the frontal cortex, cerebellum, and liver. Outer-ring activity was determined by measuring the release of radioiodide from 100,000 cpm (in the experiments with the liver 200,000 cpm) (3, 5, 3')[125I]T3 under the following conditions: in individual experiments the effects of varying pH values were investigated, thus evaluating a pH range of between 5 and 9; in other experiments, the DTT concentrations were raised to 40 and 80 mM, respectively; in a further set of experiments, the incubation times were prolonged to 120 and 180 min, respectively. Finally, the effects of the cofactors NAD, NADPH, NADH, and FAD were tested under standard conditions.

Statistics
The correlations between the different hormones and between the hormone concentrations and deiodinase activities across brain regions were calculated by Pearson’s coefficient of correlation. The tissue concentrations of hormones measured six times during a 24-h cycle were analyzed by one-way ANOVA. When significant differences were found, periodic regression analysis was performed by Fourier periodic curve fitting, using the software package COSTAT (for details see Ref. 22).

All data are given as means ± SEM and results yielding a p value of less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Hormone determination
 Results
Discussion
 References
 
RIA for rT3
Figure 1Go presents the standard curve showing the displacement of the rT3 tracer from the specific antibody by increasing concentrations of nonradioactive rT3. The rT3 standard curve was identified by the following displacement parameters: ED80 = 2.3 fmol/tube; ED50 = 10.8 fmol/tube; and ED20 = 46.1 fmol/tube. The lowest standard concentration of 1.0 fmol/tube induced an approximately 10% inhibition of rT3 binding.



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  		Figure 1. Standard curve for reverse T3. Increasing concentrations of nonradioactive reverse T3 were added to displace the binding of the [125I]-rT3 tracer to its specific antibody.

 
The abilities of various iodothyronines and iodotyrosines to displace the binding of rT3 to its specific antibody were tested in four to five different concentrations. Cross-reaction of the rT3 antibody was minimal with all compounds tested. The highest relative cross-reactivity was found for 3,3'-T2 (1.0%). L-T3 and D-T3 showed values of 0.02 and 0.032%, respectively. The results of the cross-reactivity tests for L-T4 were found to be not parallel to the rT3 standard curve, varying between 0.1 and 0.025%.

Inter and intraassay coefficients of variation (CVs) for tissue samples were determined measuring 6 samples in three successive assays (interassay test) and 30 different samples in triplicate in the same assay (intraassay test). The CVs of intraassay reproducibility ranged from 4.1–6.3%, the interassay CVs from 9.2–11.7%. The measurements were performed using samples which caused approximately 50% inhibition of rT3 antibody binding.

HPLC purification and separation of iodothyronines
A series of experiments was performed to process only radioactively labeled hormones diluted in the eluent through the HPLC column, dry them by vacuum centrifugation, and then process them through the HPLC column again to investigate whether deiodination occurs during the HPLC and drying procedures. The results show that all hormones remained stable under the conditions applied. No radioactive iodine was detected in the second HPLC run. In further experiments known amounts of cold hormones were processed through the chromatographic column and quantified by RIA. Approximately 80–90% of the known concentration of each hormone were measured in the RIA, i.e. the process of HPLC separation and drying of the samples results in a loss of between 10 and 20%. The final recovery rates of the tissue homogenates ranged from 60–80% for T3 and from 40–60% for T4. Additional losses were caused by filtering the hormones once (for small areas of the brain) and twice (large brain regions) after the extraction procedure (see above).

The blanks, which were processed simultaneously with the tissue samples by extraction and passage through the HPLC column, usually yielded nonmeasurable values in the RIA, or, in some exceptional cases, very low values, always below 1 femtomol/tube.

In preliminary experiments, we had found that sampling the HPLC fractions in plastic tubes yielded relatively low and inconsistent recovery values. This was due to the fact that it was not possible to resuspend the samples efficiently in the RIA buffers when they were dried in plastic tubes. The HPLC fractions were therefore always collected and dried in glass tubes.

Neither the addition of phloretin, IOP, or PTU before homogenization, nor the addition of these compounds or different detergents such as Triton-X during the extraction resulted in higher concentrations of any of the thyroid hormones under investigation (data not shown).

Thyroid hormone concentrations in homogenates
Figure 2Go shows the distribution of 3,5-T2, 3,3'-T2 and rT3 in 12 areas of the brain and the pituitary and liver of the rat. 3,5-T2 was measurable in 6 out of the 12 brain regions and the liver but was not detectable in the pituitary glands. The 3,5-T2 brain concentrations ranged from 16.6 ± 1.9 fmol/g in the cerebellum to 45.8 ± 1.5 fmol/g in the amygdala.



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  		Figure 2. Concentrations of 3,5-T2, 3,3'-T2 and rT3 in brain regions, the pituitary gland and liver of the rat. Cf, Frontal cortex; Cp, parieto-occipital, cortex; Str, striatum; Lf, limbic forebrain; Sep, septum; Amy, amygdala; Hip, hippocampus; Mid, midbrain; Hyp, hypothalamus; Ob, olfactory bulb; Crb, crebellum; Med, pons/medulla; Pit, pituitary; Liv, liver; n.d., not detectable; n.m., not measured.

 
3,3'-T2 was detectable in 10 of 12 brain regions. The concentrations ranged from 15.2 ± 3.8 fmol/g in the medulla to 197.3 ± 12.4 fmol/g in the cerebellum. 3,3'-T2 was not detectable in the midbrain, hypothalamus, pituitary, or liver.

The rT3 concentrations were detectable in all regions of the brain investigated except the olfactory bulb and also in the liver, but not in the pituitary gland. Across the brain, regions rT3 concentrations varied between 36.2 ± 3.1 fmol/g in the cerebellum to 255 ± 91 fmol/g in the septum.

The concentrations of T3S and 3,3'-T2S are shown in Fig. 3Go. T3S was not measurable in any of the brain regions apart from the hypothalamus (concentration 95 ± 11.4 fmol/g). The level of T3S in the liver was 164 ±24 fmol/g.



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  		Figure 3. Concentrations of T3 sulfate and 3,3'-T2 sulfate in brain regions, the pituitary gland and liver of the rat. For an explanation of the abbreviations see Fig. 2Go. n.d., Not detectable. n.m., Not measured.

 
3,3'-T2S was measurable in all areas of the brain under investigation but not in the liver or pituitary gland. The brain concentrations of 3,3'-T2S ranged from 52 ± 5.1 to 588 ± 104 fmol/g in the striatum and septum, respectively.

Figure 4Go shows that T4 was measurable in all 12 regions of the brain as well as in the pituitary gland and liver. The concentrations ranged between 1051 ± 82 fmol/g in the frontal cortex to 6255 ± 958 fmol/g in the olfactory bulb. The levels of T4 in the pituitary and liver were 27.7 ± 7.5 pmol/g and 71.0 ± 9.4 pmol/g, respectively.



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  		Figure 4. Concentrations of T4 and T3 in brain regions, the pituitary gland and liver of the rat. For an explanation of the abbreviations see Fig. 2Go.

 
The concentrations of T3 were also measurable in all brain areas investigated as well as in the pituitary glands and livers. They ranged from 792 ± 60 fmol/g in the amygdala to 2821 ± 150 fmol/g in the midbrain. A T3 concentration of 3980 ± 95 fmol/g was found in the pituitary. In the liver, we measured 2106 ± 170 fmol/g.

Comparison of the concentrations of T4, T3, rT3, T3S, 3,3'-T2S, 3,3'-T2 and 3,5-T2 reveals that in the brain the concentrations of T4 were roughly equal to or slightly higher than those of T3, whereas the levels of the other 5 hormones were approximately 10- to 20-fold lower (Figs. 2–4GoGoGo). In the liver, the ratio between the levels of T3 and T4 was approximately 1:35.

Mitochondrial concentrations of iodothyronines
The concentrations of 3,3'-T2 and 3,5-T2 were measured in the mitochondrial fractions of the amygdala and parieto-occipital cortex. Neither hormone was detectable in the mitochondrial fraction of the amygdala or parieto-occipital cortex in four pools of two brain regions each. Thus, in a second study 3 x 12 amygdalas and 3 x 6 parieto-occipital cortices were pooled to clarify whether our failure to detect 3,5-T2 and 3,3'-T2 in the mitochondrial fractions of two of the brain regions under investigation was due to insufficient sensitivity of the RIA employed. Again, neither 3,3'-T2 or 3,5-T2 was detectable, even in the mitochondrial pools from 12 amygdalas and 6 parietooccipital cortices (data not shown). For comparison, T3 and T4 were also measured in the mitochondrial fractions of two amygdalas. While T4 was not detectable, T3 was measurable in concentrations of 5.07 ± 0.6 fmol/mg protein (full data not shown).

Deiodinase activities
The distribution of D1 activity across brain regions and in the liver and pituitary gland is shown in Fig. 5Go. The activity of this enzyme ranged between 28 ±1.2 fmol/mg protein·h in the midbrain to 480 ± 55 fmol/mg protein·h in the hippocampus, reached 147 ± 15 fmol/mg protein·h in the pituitary and 235 ± 20 pmol/mg protein·h in the liver.



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  		Figure 5. Activities of D1, D2, D3 in regions of the brain, the pituitary gland and liver of the rat. For an explanation of the abbreviations, see Fig. 2Go. *, fmol iodide/mg protein·min. **, pmol iodide/mg protein/h.

 
The distribution of the activity of D2 is also shown in Fig. 5Go. The values ranged between 28 ± 2.5 fmol/mg protein/h in the midbrain to 222 ± 20 fmol/mg protein/h in the cerebellum.

The activities of the D3 isoenzyme ranged between 720 fmol/mg protein·h in the midbrain to 16,320 fmol/mg protein/h in the amygdala.

Correlations between tissue concentrations of thyroid hormones and deiodinase activities across brain regions
Among all calculations of the correlations between the deiodinase activities and hormone concentrations across the brain regions, there was only one significant result: the T3 concentrations were negatively correlated to the D3 activities (r = -0.752, P < 0.019). The correlations between T3 concentrations and D1 and D2 deiodinase activities were much weaker (P = 0.215 and 0.211, respectively).

Calculations of the relationship between the hormones showed a highly significant correlation between rT3 and 3,3'-T2S (r = 0.924, P = 0.0001).

Investigation of a possible outer-ring deiodination pathway of T3
The extensive study of a possible outer-ring deiodination under the different conditions described in Materials and Methods failed to reveal any deiodination of T3.

Circadian variation of 3,5-T2, T3 and T4
The circadian variations of the tissue concentrations of T3, T4, and 3,5-T2 in the cerebellum, midbrain, and liver are shown in Figs. 6–8GoGoGo. The tissue concentrations of all three hormones displayed significant circadian variations in all three areas. In particular, the tissue levels of 3,5-T2 showed significant daily variations in the liver (F = 4.9, P < 0.002), in the midbrain (F = 3.03, P < 0.02), and in the cerebellum (F = 3.2, P < 0.01). A close synchronizm is evident in all three tissues and particularly between T3 and 3,5-T2. However, whereas in the cerebellum the concentrations of all hormones increased between 2400 h and 0800 h in the cerebellum, in the midbrain the increases in 3,5-T2 and T3 were seen between 0800 h and 1200 h and ran contrary to the T4 levels, which were highest during the dark (active) phase and fell between 0400 h and 0800 h. In the liver, the highest levels of all hormones were observed at between 1200 h and 1600 h, and the diurnal patterns of all three hormones were closely parallel.



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  		Figure 6. Concentrations of T4, T3 and 3,5-T2 during a 24-h cycle in the midbrain of the rat.

 


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  		Figure 7. Concentrations of T4, T3 and 3,5-T2 during a 24-h cycle in the cerebellum of the rat.

 


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  		Figure 8. Concentrations of T4, T3, and 3,5-T2 during a 24-h cycle in the liver of the rat.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Hormone determination
 Results
 Discussion
References
 
In our study, we purified and separated tissue extracts by HPLC, replacing the purification by column chromatography as previously described (Ref. 20) and also applied by our own group (e.g. Refs. 18, 19, 22). The advantages of this method are at least 2-fold. First, several iodothyronines of low abundance from the same tissue can be separated and quantified, and second, the samples are purified much more reliably, yielding more consistent results than after purification by column chromatography, at least in our experience. We had previously already reported that the original extraction method, which consisted of a complicated procedure of extraction and back-extraction steps to remove lipids, can be replaced by a simple extraction with methanol (25). The tissue concentrations of T3 and T4 of the different methods are highly consistent. Morreale de Escobar’s group (20) reported concentrations of T3 and T4 in adult male rat brain of 1.70 and 3.47 ng/g, respectively. This is equal to approximately 2.6 pmol/g for T3 and 4.4 pmol/g for T4. Figure 4Go shows that the mean tissue levels of T3 found in our study ranged between 0.8 and 3 pmol/g and those for T4 between 1 and 6.2 pmol/g. These data demonstrate that both the simplified extraction procedure and the method of separation and purification by HPLC yield results comparable to those obtained by the method developed by Morreale de Escobar. In another study, Escobar-Morreale et al. (35) reported approximately 2- to 3-fold higher concentrations of T3 and T4 in the cerebral cortex and cerebellum of young female Wistar rats. Previous studies by our own group having applied the full extraction methods described by Morreale de Escobar (20) resulted in levels of T4 and T3 with exactly the same range as reported here. In rat frontal cortex, for example, we previously measured a mean T3 concentration of 2,300 fmol/g and a mean T4 concentration of 1,030 fmol/g tissue in control rats (36), having employed Morreale de Escobar’s method. In the present study, we found a mean T3 concentration of 2,080 fmol/g tissue and a T4 level of 1,070 fmol/g tissue in the same brain area (Fig. 4Go). The results for the two methods of extracting and purifying iodothyronines are therefore comparable and differences may be due to different rat strains, different ages, and sex (35), pretreatment modalities such as ip injections (19) or simply due to interassay variations between different laboratories and over several years. Extracting iodothyronines with methanol and separating and purifying them by HPLC is thus, in our opinion, easier to handle, allows the quantification of several iodothyronines from the same tissue and yields as reliable results as the procedure established by Morreale de Escobar.

This is the first study to have directly quantified 3,5-T2, 3,3'-T2, rT3, 3,3'-T2S, and T3S in different regions of the brain, the pituitary gland and liver of the rat. According to current knowledge, 3,5-T2 may be the most relevant of these hormones, as several independent study groups have unanimously reported physiologic effects of this hormone. In the brain, reliable detection of 3,5-T2 concentrations was possible in only 6 out of 12 areas. The levels measured were in the low femtomol range. 3,5-T2 was not even detectable in relatively large brain regions such as the frontal cortex (weight approximately 200 mg), whereas it was measurable in some smaller areas such as the amygdala (weight approximately 80 mg). These results show that our failure to detect 3,5-T2 in several brain regions was not simply due to the fact that these were the smaller areas in which we were unable to measure this hormone because the sensitivity of our RIA was too low. It would also seem unlikely that the levels of 3,5-T2 are merely derived from the blood because in this case the concentrations should be more or less correlated to the amount of tissue. There is evidently a specific distribution of 3,5-T2 across brain regions that is independent of the weights of the specific areas. The sensitivity of our 3,5-T2 RIA was in the low femtomolar range and we were reliably able to measure 2 fmol per tube (ED80 = 2.2 fmol/tube). If our RIA was not sensitive enough to detect 3,5-T2 in several brain areas such as the frontal cortex, the concentration of 3,5-T2 in these areas must therefore be in the attomolar range.

Several studies having reported physiological effects of 3,5-T2 used relatively high doses. Goglia’s group, for example, administered doses of between 1 and 10 µg 3,5-T2/100 g body weight in vivo (e.g. Refs. 5, 8, 10) and obtained significant effects at doses of 1 nmol 3,5-T2 in vitro (5). Baur et al. (9) administered 100 µg T3/kg body weight in vivo and 3–30 nmol 3,5-T2 in vitro to measure hormone effects at the pituitary level. Horst et al. (6) found in vitro effects in perfused livers after administration of 1 pmol 3,5-T2 and in vivo effects at doses of 20 µg/100 g body weight at the pituitary level (7).

These data show that the physiological effects, for example, in the pituitary gland in vitro (9), were observed at doses several orders of magnitude higher (3 nmol) than those occurring in vivo, as we did not measure 3,5-T2 at all in the pituitary and its hypothetical concentrations should therefore be in the attomolar range. Even the lowest dose administered in vivo in all these studies [1 µg/100 g body weight (10)], i.e. approximately 3 µg/rat, should result in much higher tissue concentrations of 3,5-T2 than measured under physiological conditions, although to our knowledge this has not yet been directly measured. In conclusion, our results underline the need for direct measurement, whether or not any administration of 3,5-T2, which may induce physiological effects, also lead to physiological concentrations of 3,5-T2.

We also investigated whether 3,5-T2 and 3,3'-T2 are measurable in mitochondrial fractions of rat brain regions, as mitochondrial binding of these hormones has previously been reported in the liver (21, 37) and several of the physiological effects of 3,5-T2 have been claimed to take place in the mitochondria (e.g. Ref. 38 ; for a review see Ref. 39).

Even in a pool of twelve amygdala and six parieto-occipital cortices, we were unable to detect 3,5-T2 in the mitochondrial fractions. T3 was measurable in a pool of two amygdalas in a concentration of 5.07 ± 0.6 fmol/mg protein. This may, however, reflect the presence of high affinity binding sites for T3 that have been shown to occur in mitochondria, at least, in the liver (40). These data show that there is no particular enrichment of 3,5-T2 at the mitochondria, at least, not in the brain. Even if mitochondrial binding of this hormone takes place in some regions of the brain, its concentration must be far below the sensitivity of our RIA, i.e. in the attomolar range. These results do not, however, exclude the possibility that mitochondrial binding of 3,5-T2 takes place in other tissues such as the liver (29).

A further unsolved question is the metabolic pathway by which 3,5-T2 is produced. Under our experimental conditions, at least, no outer-ring deiodination of T3 occurred. However, another study group reported production of 5–10% iodine from outer-ring labeled T3 in maternal and fetal rat cerebrocortical microsomes after incubation with 360 µg/ml microsomes (41). Further studies must finally clarify whether T3 is the direct precursor of 3,5-T2 and by which enzymatic pathway the production of 3,5-T2 is catalyzed in vivo. In the study on diurnal variations the courses of the concentrations of 3,5-T2 closely paralleled those of T3 in the cerebellum, midbrain, and liver. In the midbrain, the levels of 3,5-T2 and T3 increased, whereas those of T4 fell. This may suggest that not only T3, but also 3,5-T2 is indirectly derived from T4 via an as yet unknown metabolic pathway. In the cerebellum and liver, however, the concentrations of all these hormones changed roughly in parallel.

Diurnal variations of the serum concentrations of T4, T3, and TSH have previously been reported in rats (e.g. Ref. 22). In that study, TSH levels were higher during the day than during the night, peak values occurring at 0800 h, followed by peaks in serum levels of T4 at 1600 h and in those of T3 at 1200 h (22). However, Figs. 6Go and 7Go show that the peaks and troughs of T4 occurred at different time points in the two brain regions: between 2000 h and 0800 h in the midbrain and at 1600 h in the cerebellum. These results therefore militate against a significant contamination of the brain tissue with plasma T4. The mechanisms underlying these changes are as yet unclear and probably complex and may include variables such as diurnal variations in the serum levels of TSH and thyroid hormones, diurnal variations in enzyme activities of different tissues, as well as uptake mechanisms.

We were also unable to detect mitochondrial enrichment of 3,3'-T2, in the brain, which has been reported to occur in the liver by another study group (39).

It would seem interesting to consider whether or not the relatively low concentrations of rT3 have any relevant physiological function, e.g. inhibition of D2 activity. Obregon et al. (4) reported a 75% inhibition of cortical D2 activity in thyrectomized rats at a concentration of 533 pg/g rT3 10 min after the injection of 0.75 µg rT3. After 3 h, the inhibition was still 50% and the rT3 concentration 49 pg/g, which is not very different from the endogenous rT3 levels measured in cortical areas in the present study (~50 fmol/g). As in the study by Obregon et al., rT3 was found to be five times as potent as T4 in suppressing 5'-deiodinase activity, it would seem conceivable that rT3 is a potent modulator of brain D2 activity and T3 production in vivo. It should, however, be noted that the T4 levels are approximately 20 times as high as those of rT3, and T4 may thus have a more potent inhibiting action on D2 in vivo.

Our finding of measurable 3,3'-T2S levels across all brain areas investigated is consistent with the results of a study by Esfandiari et al. (13) who reported production of 3,3'-T2S from T3 after deiodination to 3,3'-T2 in cultured astrocytes. We failed to detect T3S in the brain (except in the hypothalamus). Hurd et al. (12) reported considerable production of T3S from T3 in an in-vitro assay of rat brain cytosol. However, they used micromolar concentrations of T3 and their results did not therefore reflect physiological conditions. Otten et al. (14) and Kaptein et al. (42) found that if 3,3'-T2 is added to rat liver cells or cytosol, respectively, 3,3'-T2S is the major metabolite produced (for a review, see Ref. 16). However, again, they employed micromolar concentrations of 3,3'-T2. We were unable to detect 3,3'-T2S in an aliquot of approximately 300 mg rat liver. We cannot, however, exclude the possibility that this hormone may be detectable if measured in whole rat liver. T3S has been observed in in vitro experiments in hepatocytes after incubation with small amounts of T3 [1 or 10 nM (15)]. Likewise in our in vivo experiments T3S was clearly detectable in the liver.

We calculated the correlations between thyroid hormone concentrations and deiodinase isoenzymes across brain regions. Although the results of such numerous correlation calculations should be interpreted with caution, they may add some knowledge worthy of mention. There was a significant negative correlation between the tissue concentrations of T3 and D3 activity across all brain areas investigated, but a much weaker correlation between T3 and D1 and D2 activities. These results indicate that D3 may be most relevant for the determination of brain concentrations of T3. This seems plausible, as D3 activity is between 20- and 105-fold higher than that of D2. Similar results were recently reported by another study group (44). There was an unexpectedly high positive correlation between 3,3'-T2S and rT3 (r = 0.925, P = 0.0001), which is unlikely to have occurred by chance. This correlation may be explained as follows. High levels of rT3 may suggest high activity of D3. If D3 is high, then T3 is rapidly converted to 3,3'-T2, which is again rapidly sulfated.

To our knowledge, the regional expression pattern of the mRNA concentrations of D2 has not yet been studied in adult rats. D3 mRNA concentrations across brain areas in a study by Tu et al. (45) do not correspond to the respective activities found in the present study. D3 mRNA, for instance, was several times higher in the hippocampus than in the cortex under euthyroid conditions, whereas the D3 activity is approx. equally high in the two areas. The surprisingly high D3 activity in the amygdala was obviously not paralleled by similarly high mRNA concentrations (45). These data indicate that posttranslational modifications may play an important role in determining deiodinase activities.

In conclusion, we developed a new HPLC-based method for purifying and separating different iodothyronines before quantification by RIA. This method permits reliable detection of five iodothyronines other than T3 and T4 in regions of the rat brain and/or rat liver. The possible physiological functions of these iodothyronines, particularly rT3 and 3,5-T2 remain to be further investigated.


   Footnotes
 
This study was supported by the Deutsche Forschungsgemeinschaft (Grant Ba 932/7-2).

Abbreviations: CV, Coefficient of variation; D1, D2, D3, type I, II, or III deiodinase; DTT, dithiothreitol; IOP, iopanoic acid; MBq, megabecquerels; NAD, ß-nicotinamide adenine dinucleotide; PTU, 6-n-propyl-2-thiouracil; rT3, reverse T3; S1, low-speed supernatant; 3,5-T2, 3,5- diiodothyronine; 3,3'T2S, 3,3'-T2 sulfate; T3S, T3 sulfate.

Received October 19, 2001.

Accepted for publication January 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Hormone determination
 Results
 Discussion
 References
 

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