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The Zn concentration in thyroid tissue and whole blood of patients with Graves' disease, thyroid cancer, and nodular goiter disease was determined using the total-reflection X-ray fluorescence method. The dependence of obtained concentrations on the clinical stage of the examined disease, histopathological grading, and kind of analyzed material (thyroid tissue and blood) was studied. The determined concentration of Zn was the lowest in the thyroid tissue of patients with thyroid cancer (23.1 microg/g) and it was the highest in the case of Graves' disease (41.7 microg/g), whereas in the blood samples, the reverse results were found (7.1 microg/g and 4.8 microg/g, respectively). The physical basis of the method used, the experimental setup, and the procedure of sample preparation are described.

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It is shown that 2-10 microM Zn2+ induces swelling of rat liver mitochondria incubated in a buffered sucrose medium either with valinomycin or with FCCP, Ca2+, ionophore A23187, oligomycin, and nigericin. This swelling was associated with the release of GSH from mitochondria. Both processes were sensitive to known inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A, and Mg2+. Mitochondrial swelling induced by Zn2+ was also inhibited by rotenone, antymycin A, N-ethylmaleimide, butylhydroxytoluene, and spermine, whereas it was stimulated by tert-butyl hydroperoxide, diamide, and monobromobimane. It did not require the addition of phosphate. The same sensitivity to pH of the mitochondrial swelling induced by Zn2+ and by phenylarsine oxide suggests the same site of the interaction, namely, thiol groups. The ability of Zn2+ to induce mitochondrial swelling gradually decreased along with its increasing concentration above 10 microM. It is concluded that micromolar Zn2+ induces the MPT presumably by the interaction with cysteinyl residues. This process is independent of the mitochondrial membrane potential.

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The oxidation of some mitochondrial sulfhydryl groups acts as an inducer of the mitochondrial membrane permeability transition. This membrane damage can be reversed by regeneration of the sulfhydryl groups. Hence the mitochondrial activities reducing disulfides are especially important for the defense against oxidative injury. The ability of isolated rat liver mitochondria to reduce disulfides was examined by the reduction of 5,5'-dithiobis-(2 nitro-benzoic acid) (DTNB), the reaction catalyzed by thioredoxin reductase and glutathione reductase. The incubation of mitochondria with DTNB induced their swelling which was prevented by EGTA, cyclosporin A, Mg2+, and pyrophosphate. The rate of DTNB reduction by mitochondria was compared with the changes in their volume and in the total content of their sulfhydryl groups. The reduction of DTNB was stimulated by cation chelators, both unphysiological and physiological, and was suppressed by up to 65% during mitochondrial swelling. In mitochondria treated with tert-butylhydroperoxide the total content of mitochondrial sulfhydryl groups decreased in the correlation with the suppression of DTNB reduction and with mitochondrial swelling. The content of nonprotein sulfhydryl groups was significantly lowered under all conditions applied. However, the protein sulfhydryl groups were preserved in the presence of EGTA or Mg2+ when the rate of DTNB reduction was relatively high and this was associated with the strong inhibition of mitochondrial swelling. It is suggested that during oxidative stress mitochondrial activities reducing disulfides are involved in the maintenance of sulfhydryl groups in mitochondria thus protecting their membranes against permeabilization.

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The oxidation of mitochondrial sulphydryl groups is known to increase the permeability of mitochondrial membranes and to be a key event in oxidative stress. Resistance to this damage is thought to involve thioredoxin reductase. In this study, the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) by a lysate of rat liver mitochondria was used to assay the mitochondrial disulphide reducing capacity. NADPH-dependent reduction of DTNB was used to distinguish enzymatic reduction from the non-enzymatic reduction. Enzymatic reduction by the mitochondrial lysate was suppressed by DTNB at concentrations exceeding 0.25 mM and by pH above 7.0. It was strongly inhibited by Zn2+ and Mn2+ (IC50 about 2.5 and 20 microM, respectively) and was more weakly inhibited by Mg2+ and Ca2+ (IC50 about 1.8 and 2.1 mM, respectively). As a consequence of inhibition by divalent cations, the reaction was stimulated by both physiological (ATP, ADP, pyrophosphate and citrate) and non-physiological (EDTA and EGTA) chelators. Reduction of insulin disulphides by the mitochondrial lysate was dependent on the presence of a divalent cation chelator during the isolation of mitochondria and during the enzyme assay. Our results suggest that stimulation of mitochondrial disulphide reducing activity by lowered pH, as well as by increased levels of ATP, ADP and citrate, has the potential to contribute to the maintenance of mitochondrial sulphydryl groups under oxidative conditions.

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