Film autoradiography identifies unique features of [125I]3,3'5'-(reverse) triiodothyronine transport from blood to brain.

1. Steady-state iodothyronine profiles in plasma are composed of thyroid gland-synthesized hormones (mainly thyroxine) and tissue iodothyronine metabolites (mainly triiodothyronine and reverse triiodothyronine) that have entered the bloodstream. The hormones circulate in noncovalently bound complexes with a panoply of carrier proteins. Transthyretin (TTR), the major high-affinity thyroid hormone binding protein in rat plasma, is formed in the liver. It is also actively and independently synthesized in choroid plexus, where its function as a chaperone of thyroid hormones from bloodstream to cerebrospinal fluid (CSF) is undergoing close scrutiny by several groups of investigators. Because TTR has high-affinity binding sites for both thyroxine and retinol binding protein, its potential role as a mediator of combined thyroid hormone and retinoic acid availability in brain is of further interest. 2. While they are in the free state relative to their binding proteins, iodothyronines in the cerebral circulation are putatively subject to transport across both the blood-brain barrier (BBB) and choroid plexus CSF barrier (CSFB) before entering the brain. Previous autoradiographic studies had already indicated that after intravenous administration the transport mechanisms governing thyroxine and triiodothyronine entry into brain were probably similar, whereas those for reverse triiodothyronine were very different, although the basis for the difference was not established at that time. Intense labeling seen over brain ventricles after intravenous administration of all three iodothyronines suggested that all were subject to transport across the CSFB. 3. To evaluate the role of the BBB and CSFB in determining iodothyronine access to brain parenchyma, autoradiograms prepared after intravenous administration of [125I]-labeled hormones (revealing results of transport across both barriers) were compared with those prepared after intrathecal (icv) hormone injection (reflecting only their capacity to penetrate into the brain after successfully navigating the CSFB). 4. Those studies revealed that thyroxine and triiodothyronine were mainly transported across the BBB. They shared with reverse triiodothyronine a generally similar, limited pattern of penetration from CSF into the brain, with circumventricular organs likely to be the main recipients of iodothyronines (with or without retinol) transported across the CSFB. 5. Analysis of all of the images obtained after intravenous and icv hormone administration clarified the basis for the unique distribution of intravenously injected reverse triiodothyronine. The hormone is excluded by the BBB but may be subject to limited penetration into brain parenchyma via the CSF. 6. Overall the observations single out reverse triiodothyronine as the iodothyronine showing the most distinctive as well as the most limited pattern of transport from blood to brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Desmethylimipramine, a potent inhibitor of synaptosomal norepinephrine uptake, has diverse effects on thyroid hormone processing in rat brain. II. Effect on in vivo 5'-deiodination of [125I]thyroxine.

We have studied the effects of desmethylimipramine (DMI), a tricyclic antidepressant, on thyroid hormone (TH) handling in rat brain in an effort to discover a pharmacological basis for reported interactions between TH, affective disorders and psychotropic drugs. An acute dose of DMI has been used in order to determine the primary effects of the drug in brain without perturbations from secondary effects. Recently we have reported that a single dose of DMI significantly decreases brain uptake of both [125I]thyroxine (T4) and [125I]3,3',5-triiodothyronine (T3) across the spectrum of thyroid states from hypothyroid (HYPO) to euthyroid (EU) to T4-induced hyperthyroid (HYPER). To investigate further the effects of DMI on brain processing of TH, we have measured effects of the drug on in vivo rates of T4 to T3 conversion in a series of experiments in which DMI (25 mg/kg) was given to HYPO, EU and HYPER male rats in conjunction with i.v. [125I]T4. Decreased in vivo conversion ratios (T3/T4 ratios) suggest that acute DMI treatment causes a significant decrease in 5'-deiodinase activity in balance of brain (but not cerebellum) in all DMI treated rats as compared to their saline treated controls (ANOVA, P < 0.0001). For assurance that reduced T3/T4 in DMI treated rat brain is not the result of DMI enhancement of 5-deiodination of T3 or T4, the effect of DMI on concentrations of labeled I-, rT3, and T2 (3,3'- and 3',5'-) was also observed. In no case was there a significant increase in any metabolite in DMI treated rats for any tissue studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Desmethylimipramine, a potent inhibitor of synaptosomal norepinephrine uptake, has diverse effects on thyroid hormone processing in rat brain. I. Effects on in vivo uptake of 125I-labeled thyroid hormones in rat brain.

Several lines of evidence point to an interaction between amine uptake inhibitors (tricyclic antidepressants) and thyroid hormones. To examine this issue under conditions which would minimize secondary effects of drug treatment, desmethylimipramine (DMI), a highly specific norepinephrine uptake inhibitor, was given acutely as a single i.p. dose one hour before i.v. [125I]triiodothyronine (T3*) or [125I]thyroxine (T4*). Tissues were analysed after rat decapitation at 3, 5, 10, and 20 min intervals thereafter. DMI had a small but significant inhibitory effect on the brain uptake of both T3* (7.4%) and T4* (19%) over their respective 20-min time courses as indicated by two-way ANOVA. To examine the drug response further and to determine the effect of thyroid status on the response, hypothyroid (HYPO) and T4-induced hyperthyroid (HYPER) rats, were given i.v. T3* and, 5 min later, i.p. DMI or saline. They were killed 3 h later and tissue analysed. Because DMI effects on T4* uptake could not be evaluated over a 3 h period without blocking T4* to T3* conversion, sodium ipodate (60 mg/kg) was given in 2 doses before i.v. T4*. Under these conditions, DMI significantly reduced brain concentrations of the administered T3* and T4* in HYPO (15% and 19%) and in HYPER rats (13% and 25%). These results suggest that, as it does in the case of norepinephrine, DMI blocks the uptake site for T3 and T4 in rat brain. No information is available regarding the relationship, if any, between the thyroid hormone and norepinephrine uptake sites.

Determination of saccharides in biological materials by high-performance anion-exchange chromatography with pulsed amperometric detection.

High-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) under alkaline conditions (pH 9-13) separates aminosaccharides, neutral saccharides and glycuronic acids based upon their molecular size, saccharide composition and glycosidic linkages. Carbohydrates were extracted by utilizing 0.5 M H2SO4 (neutral monosaccharides), 0.25 M H2SO4 coupled with enzyme catalysis (glycuronic acids) and 3 M H2SO4 (aminosaccharides). Solid-phase extraction with strong cation and strong anion resins was used to partition the cationic aminosaccharides and anionic glycuronic acids and to deionize acid extracts for neutral saccharides. Separation was conducted on a medium-capacity anion-exchange column (36 mequiv.) utilizing sodium hydroxide (5-200 mM and sodium acetate (0-250 mM) as the mobile phase. The saccharides were detected by oxidation at a gold working electrode with triple-pulsed amperometry. HPAEC-PAD was found superior to high-performance liquid chromatography with refractive index (RI) detection for neutral monosaccharides and aminosaccharides and to low-wavelength UV detection for glycuronic acids in terms of resolution and sensitivity. HPAEC-PAD was not subject to interferences as was the case for low UV detection (210 nm) or RI analyses and was highly selective for mono- and aminosaccharides and glycuronic acids. The use of HPAEC-PAD was applied for the determination of the saccharide composition of organic materials (plant residues, animal wastes and sewage sludge), microbial polymers and soil.

Determination of aminosaccharides by high-performance anion-exchange chromatography with pulsed amperometric detection.

High-performance anion-exchange chromatography (HPAC) was used for the determination of aminosaccharides in microbial polymers, chitin, animal waste, sewage sludge, plant residues and soil. The aminosaccharides, galactosamine, mannosamine and glucosamine were separated on a strong anion-exchange column with 1OmM sodium hydroxide as the eluent and determined by pulsed amperometric detection (PAD). The HPAC-PAD methodology was compared with high-performance liquid chromatography (HPLC) with refractive index detection (RI) in terms of selectivity and sensitivity for aminosaccharides. The results indicate that HPAC-PAD required less sample preparation, and was more precise and nearly two orders of magnitude more sensitive than HPLC-RI. HPAC-PAD was not subject to matrix interferences and was highly selective for aminosaccharides. More than 3% of the total nitrogen in alfalfa, and 20% of that in straw, was found to be present as aminosaccharides.