Combined effects of estrogen deficiency and cadmium exposure on calcified hard tissues: animal model relating to itai-itai disease in postmenopausal women.

Using ovariectomized rats as a model of postmenopausal women, we studied the effects of estrogen (Es) deficiency and in combination with cadmium (Cd) exposure on the calcified hard tissues related to the development of itai-itai disease. Es deficiency suppressed the synthesis of carbonic anhydrase required for the crystal nucleation process, causing the crystal structure defects in the tooth enamel. Regarding the combined effects of Es deficiency and Cd exposure on the bone, in which rats were given drinking water containing Cd ions, soft X-ray radiography revealed a development of labyrinthine pattern in the calvaria, and micro-computed tomography demonstrated the declining trabecular architecture of the tibia, suggesting Cd-induced osteoporotic change. Further, electron microscopy showed the increase of amorphous minerals in the calvaria. In conclusion, the combined effects of Es deficiency and Cd exposure can be responsible for accelerating the declining bone strength together with the crystal structure defects resulting in the preferential occurrence of itai-itai disease in postmenopausal women.(Communicated by Tatsuo SUDA, M.J.A.).

Electron microscopy of octacalcium phosphate in the dental calculus.

The purpose of this study was to morphologically demonstrate the presence of octacalcium phosphate in the dental calculus by judging from the crystal lattice image and its rapid transformation into apatite crystal, as part of our serial studies on biomineral products. We also aimed to confirm whether the physical properties of octacalcium phosphate are identical with those of the central dark lines observed in crystals of ordinary calcifying hard tissues. Electron micrographs showed that crystals of various sizes form in the dental calculus. The formation of each crystal seemed to be closely associated with the organic substance, possibly originating from degenerated microorganisms at the calcification front. Many crystals had an 8.2-A lattice interval, similar to that of an apatite crystal. Furthermore, some crystals clearly revealed an 18.7-A lattice interval and were vulnerable to electron bombardment. After electron beam exposure, this lattice interval was quickly altered to about half (i.e. 8.2 A), indicating structural conversion. Consequently, a number of apatite crystals in the dental calculus are possibly created by a conversion mechanism involving an octacalcium phosphate intermediate. However, we also concluded that the calcification process in the dental calculus is not similar to that of ordinary calcifying hard tissues.

Mechanism of cadmium induced crystal defects in developing rat tooth enamel.

It is well known that exposure to environmental cadmium causes itai-itai (ouch-ouch) disease. However, the exact mechanism underlying this bone disease remains unresolved. By focusing on the calcification mechanism, we examined developing tooth enamel in rats exposed to cadmium to test the hypothesis that cadmium exposure may cause defects in crystal formation. Electron microscopy revealed the presence of perforated crystals in developing tooth enamel, indicating that the process of crystal nucleation may have been interrupted by cadmium exposure. Furthermore, biochemical analyses revealed that the catalytic activity of carbonic anhydrase in the immature enamel matrix declined remarkably despite the fact that quantitative reduction of this enzyme was insignificant, suggesting that the decline of catalytic activity may have resulted from the replacement of zinc with cadmium ions. Therefore, we concluded that the poor catalytic activity of cadmium-binding carbonic anhydrase might hinder the nucleation process, leading to an impairment in mineralization that causes itai-itai disease.

Determination of taltirelin, a new stable thyrotropin-releasing hormone analogue, in human plasma by high-performance liquid chromatography turbo-ionspray ionization tandem mass spectrometry.

A rapid, selective and sensitive assay of taltirelin, a novel thyrotropin-releasing hormone analogue, in human plasma has been developed. This method is based on a rapid sample preparation and high-performance liquid chromatography (HPLC) turbo-ionspray ionization tandem mass spectrometry (MS-MS). Analytes were purified from human plasma by SPE cartridge and separated by gradient HPLC. Turbo-ionspray ionization and MS-MS analyses were carried out by PE-Sciex API 3000 tandem mass spectrometer. Taltirelin was separated from its metabolite (acid form) on a semi-micro ODS column in methanol - 0.1% (v/v) formic acid. The selected reaction monitoring by precursor-->product ion combination of m/z 406-->264, was used for determination of taltirelin. The linearity was confirmed in the concentration range of 17-4137 pg/ml in human plasma, and the precision of this assay, expressed as a relative deviation, was less than 9.8% over the entire concentration range with adequate assay accuracy. The results obtained by the HPLC-MS-MS method correlated well with those of the radioimmunoassay method reported previously. Therefore, the HPLC-MS-MS method is useful for the determination of taltirelin with sufficient selectivity and sensitivity on pharmacokinetic studies in human.

Fluvastatin ameliorates the hyperhomocysteinemia-induced endothelial dysfunction: the antioxidative properties of fluvastatin.

BACKGROUND: Hyperhomocysteinemia induces vascular endothelial dysfunction, contributing to a predisposition to the onset and/or progression of atherosclerosis. The major mechanism suggested for the adverse effect of homocysteine on vascular function seems to involve oxidative stress. Thus, we hypothesized that the administration of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor fluvastatin, which is experimentally demonstrated to have antioxidative properties as one of its pleiotropic effects, is a useful strategy for eliminating the detrimental events induced by hyperhomocysteinemia. METHODS AND RESULTS: In diet-induced hyperhomocysteinemic rats, we estimated oxidative stress and assessed endothelium-dependent vasodilatation. Hyperhomocysteinemia induced significant increases in urinary 8-isoprostaglandin F2alpha-III excretion and vascular superoxide generation, and impaired endothelium-dependent vasodilatation. Additional oral administration of the antioxidant fluvastatin or vitamin E, which normalized increased oxidative stress induced by hyperhomocysteinemia, ameliorated endothelial dysfunction. CONCLUSIONS: Hyperhomocysteinemia, even mild to moderate, induces endothelial dysfunction through its oxidative effect. The antioxidant fluvastatin was able to cancel out the oxidative stress induced by hyperhomocysteinemia and ameliorate endothelial dysfunction. Clinical use of fluvastatin might be a potent strategy for eliminating the detrimental events induced by hyperhomocysteinemia as well as hyperlipidemia. In addition to lowering homocysteine by means of folate supplementation, administration of the antioxidants is expected to be a potentially effective anti-homocysteine therapy.

Preservation of endothelial function by the HMG-CoA reductase inhibitor fluvastatin through its lipid-lowering independent antioxidant properties in atherosclerotic rabbits.

Recent evidence suggests that the beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on entothelial function and cardiovascular ischemic events may be attributed not only to their lipid-lowering effects but also to cholesterol-lowering independent (direct) effects on the atherosclerotic vessel wall. This study was designed to test the hypothesis that fluvastatin (Flu) preserves the endothelial function by its cholesterol-lowering independent actions. Rabbits were fed a 0.5% high-cholesterol (HC) diet for 12 weeks (progression phase) and then fed the HC diet either containing or not containing Flu 2 mg/kg/day for an additional 8 weeks (treatment phase). Rabbits fed a normal diet were used as controls. Plasma total and low-density lipoprotein cholesterol concentrations did not differ during the treatment phase: Endothelium-dependent/NO-mediated relaxation (acetylcholine and A23187) was impaired in the HC diet group, whereas it was preserved in the HC plus Flu treatment group. The endothelium-independent relaxation (sodium nitroprusside) was similar between the three groups. Interestingly, aortic oxidative stress (lipid peroxides and isoprostane F(2alpha)-III contents) and NADPH oxidase component (p22phox and gp91phox) mRNA expression were increased in the HC group but not in the HC plus Flu group. The A23187-induced nitric oxide production from the aorta was increased in both HC and HC plus Flu groups. There was no significant difference in tissue endothelial-type nitric oxide synthase mRNA expression. Plaque area and intimal thickening of the aorta were significantly lowered in the HC plus Flu group. Flu treatment preserved the endothelial function associated with the decrease in markers of oxidative stress in this experiment. These beneficial endothelial effects of Flu are likely to occur independently of plasma lipid concentrations and to be mediated by its antioxidant action.