Inhibition of iodothyronine deiodinase by phenolphthalein dyes: structure--activity relationship.
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Microspheres containing poly(acrylic acid) and beta-cyclodextrin or maltose were prepared by a w/o solvent evaporation technique. The dispersed aqueous phase contained poly(acrylic acid) (800 mg) and beta-cyclodextrin or maltose (0, 200 or 800 mg). Food-grade olive oil was the continuous phase. Microsphere particle size was consistently between 15 and 25 microm, and carbohydrate content was in good agreement with that added to the dispersed phase in all cases. Two dyes, phenolphthalein and rhodamine B, having different solubility characteristics and strengths of association with beta-cyclodextrin, were selected for loading and in vitro release studies. Microspheres were loaded by soaking in a saturated propan-2-ol solution of the appropriate dye (6 h). Microsphere dye content ranged between 2.8 and 4.8 mg/g microspheres for phenolphthalein and between 2.2 and 3.7 mg/g for rhodamine B. Release studies were performed in phosphate buffer (pH 7.4; 37 degrees C). No difference in the release profile of either dye was observed between microspheres. The failure of microspheres containing beta-cyclodextrin in particular, to alter the in vitro release kinetics of either dye may be due to a number of factors and include: (i) limited cross-linking giving rise to a the rapid hydration of the polymer matrix; (ii) perturbation of the dye-beta-cyclodextrin complex by oil and/or organic solvent residues; and (iii) conformational changes/steric hindrance of the beta-cyclodextrin cavity (due to its covalent binding with PAA) resulting in a reduction in its ability to form inclusion complexes.
The efflux of [35S]sulphate from the lumen of the proximal renal tubule into tubular cells of rats was measured by the stop-flow tubular-lumen microperfusion technique. The transport parameters obtained and the apparent Ki values of competing substrates were compared with those of the contraluminal influx of [35S]-sulphate from the interstitium into tubular cells. For the luminal sulphate efflux a Km(l, SO4(2-)) of 0.8 mmol/l and a Jmax(l, SO4(2-)) of 0.2 pmol s-1 cm-1 were found. The corresponding contraluminal values were Km(cl,SO4(2-)) 1.4 mmol/l and Jmax(cl,SO4(2-)) 1.2 pmol s-1 cm-1. Omission of Na+ from the perfusates reduced the luminal efflux of sulphate by 83%, while the contraluminal influx of sulphate was not changed. Increase in HCO3- concentration inhibited both luminal efflux and contraluminal influx of sulphate, while a change of pH from 6.0 to 8.0 was without effect. Comparing the apparent Ki(SO4(2-)) values for luminal and contraluminal sulphate transport, a relationship close to 1:1 was seen for some inorganic substrates with tetrahedral molecular structure (thiosulphate, sulphate, molybdate and selenate). The same holds for phosphate, while for oxalate the contraluminal Ki(SO4(2-)) value was lower than the luminal one (1.2 and 4.5 mmol/l). Some of the dicarboxylates and disulphonates tested show the same affinity to the luminal Na(+)-dependent sulphate transporter and the contraluminal sulphate exchange system, whereas most of the benzene carboxylate and benzenesulphonate derivatives tested exhibit higher luminal than contraluminal Ki values. The inhibitory potency increased with rising numbers of substituents on the benzene ring. This effect was more pronounced for the contraluminal sulphate transporter. In general, only disulphonates and analogues as well as similarly structured compounds (5-sulphosalicylate, 2-hydroxy-5-nitrobenzenesulphonate, eosine-5-isothiocyanate) have a good inhibitory potency toward the luminal sulphate transporter [apparent Ki 0.9-3.1 mmol/l]. All the tested sulphamoyl and phenoxy diuretics, and fluorescein and phenolphthalein dyes showed no or a smaller inhibitory potency to the luminal sulphate transport system than to the contraluminal. The most effective inhibitors of both sulphate transport systems are 8-anilino-1-naphthalenesulphonate, orange G, and H2-DIDS. The data indicate that the Na(+)-dependent luminal and the Na(+)-independent contraluminal sulphate transport systems accommodate a similar spectrum of anionic substrates, whereby the inhibitory potency against the luminal Na(+)-dependent sulphate transport system is identical or smaller than against the contraluminal transporter.
This study was performed to investigate whether mucosal diamine oxidase activity could be assessed by measuring expired 14CO2 after oral administration of 14C-putrescine. Immediately after giving 5 mu Ci of 14C-putrescine, the 14CO2 was collected at 1-h intervals for 8 h into a vial containing 1 ml of 10 mM hyamine hydroxide, 2 ml of ethanol, and an appropriate amount of phenolphthalein dye. The expired 14CO2 caused the color to disappear. The amount of 14CO2, determined by scintillation counting, reached a maximum 1 h after 14C-putrescine administration, and gradually decreased thereafter. A positive correlation between the mucosal diamine oxidase activity and the maximal expired 14CO2 value was obtained. There is no doubt that this test can be used to easily detect mucosal DAO activity and avoid the necessity of mucosal biopsy.
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In order to evaluate the specificity for the contraluminal sulfate transport system the inhibitory potency of phenol- and sulfonphthaleins, of sulfamoyl-compounds (diuretics) as well as diphenylamine-2-carboxylates (Cl- channel blockers) on the 35SO4(2-) influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 1) Phenolsulfonphthalein (phenol-red) inhibited with an app. Ki-value of 1.7 mmol/l, while analogs which had additional Br-atoms in position 3 and/or 5, i.e. bromphenol-blue, bromcresol-purple and bromcresol-green, inhibited with an apparent Ki of 0.1 and 0.5 mmol/l respectively. 2) Phenolphthalein and tetrabromphenolphthalein did not inhibit, while the disulfonate dyes bromsulfalein, fuchsin acid and indigocarmine inhibited with a Ki between approximately equal to 1 and 3 mmol/l. The highest inhibitory potency in this class of compounds was seen with orange G (app. Ki 0.07 mmol/l). The monosulfonate dyes tested, fluorescein-sulfonate and orange I inhibited moderately with an app. Ki of approximately equal to mmol/l. 3) The 3-sulfamoyl compounds inhibited to a varying degree, when they had a neighbouring -NH-group (furylmethylamino-group), i.e. in position 6 to the COOH or SO3H-group, or when they had a phenoxy-group in position 4. 4) 4-sulfamoylbenzoate and the related compounds probenecid, acetazolamide and hydrochlorothiazide inhibited with an app. Ki between 4 and 7 mmol/l. 5) All diphenylamine-2-carboxylate analogs inhibited with an app. Ki between 3 and 5 mmol/l, even when the -NH-group was replaced by an = O-group or the benzene ring was replaced by a pyrimidine ring, but not when it was replaced by a thiophen ring.(ABSTRACT TRUNCATED AT 250 WORDS)
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The uterus of the pregnant spiny dogfish, Squalus acanthias, can convert phenol red into a new purplish-blue dye. Evidence shows that the new dye is bromophenol blue. This is the first example of biological bromination that has been observed in a vertebrate.
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