Iron-based phosphorus chelator: Risk of iron deposition and action on bone metabolism in uremic rats

Phosphate chelators are frequently used in patients with chronic kidney disease (CKD). New iron-based chelators remain understudied and offer a promising therapeutic option for the control of bone and mineral disorders of chronic kidney disease (BMD-CKD). We assessed the effect of the phosphorus chelator, chitosan-iron III (CH-FeCl), compared to calcium carbonate (CaCO₃) in BMD-CKD and the potential iron overload in uremic rats. Thirty-two animals were divided into four groups, namely the control, CKD, CKD/CH-FeCl, and CKD/CaCO₃ groups. CKD was induced by adding 0.75% (4 weeks) and 0.1% (3 weeks) adenine to the diet. The chelators were administered from week 3 through week 7. The renal function, BMD-CKD markers, and histomorphometry of the femur were assessed at week 7. The CKD group showed a significant increase in creatinine (83.9 ± 18.6 vs. 41.5 ± 22.1 µmol/L; P = 0.001), phosphate (3.5 ± 0.8 vs. 2.2 ± 0.2 mmol/L; P = 0.001), fractional excretion of phosphorus (FEP) (0.71 ± 0.2 vs. 0.2 ± 0.17; P = 0.0001), and FGF23 (81.36 ± 37.16 pg/mL vs. 7.42 ± 1.96; P = 0.011) compared to the control group. There was no accumulation of serum or bone iron after the use of CH-FeCl. The use of chelators reduced the FEP (control: 0.71 ± 0.20; CKD/CH-FeCl: 0.40 ± 0.16; CKD/CaCO₃ 0.34 ± 0.15; P = 0.001), without changes in the serum FGF23 and parathyroid hormone levels. Histomorphometry revealed the presence of bone disease with high remodeling in the uremic animals without changes with the use of chelators. The CH-FeCl chelator was efficient in reducing the FEP without iron accumulation, thereby paving the way for the use of this class of chelators in clinical settings in the future.     

Effect of metal substitution in copper amine oxidase from lentil seedlings

 The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k _c value is different from that of native or Cu-fully-reconstituted enzyme, while K _m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism. Received: 5 March 1999 / Accepted: 22 July 1999     

The metal function in the reactions of bovine serum amine oxidase with substrates and hydrazine inhibitors

 Bovine serum amine oxidase (BSAO) reacts with 2-hydrazinopyridine, which binds the organic cofactor 2,4,5-trihydroxyphenylalanine quinone, forming a band at 435 nm. The band shifts to 526 nm around 60  °C, to 415 nm upon denaturation, but only shifts to 429 nm upon Cu²⁺ depletion. Its wavelength and intensity suggest that the adduct has the azo conformation, whilst the same adduct of crystallineEscherichia coli amine oxidase (ECAO) shows the hydrazone conformation in the X-ray structure. The steady state kinetics of aminomethyl- and aminoethylpyridines confirm that the formation of the product Schiff base, analogous to the azo form of the 2-hydrazinopyridine adduct, is not hindered in solution. The structural stability of the adduct in the absence of Cu²⁺ is taken to imply hydrogen bonding of the pyridyl nitrogen to a conserved aspartate, as in the ECAO adduct. Thus the ECAO adduct provides a good model for a transient intermediate leading to formation of the BSAO azo adduct. On the basis of this model and of the catalytic competence of Co²⁺-substituted BSAO, confirmed by the present data, a catalytic reaction scheme is proposed. Received: 2 December 1998 / Accepted: 22 March 1999     

Mechanistic insight into the catechol oxidase activity by a biomimetic dinuclear copper complex

The biomimetic catalytic oxidation of 3,5-di-tert-butylcatechol by the dicopper(II) complex of the ligand ,-bis{bis[1-(1-methyl-2-benzimidazolyl)methyl]amino}-m-xylene in the presence of dioxygen has been investigated as a function of temperature and pH in a mixed aqueous/organic solvent. The catalytic cycle occurs in two steps, the first step being faster than the second step. In the first step, one molecule of catechol is oxidized by the dicopper(II) complex, and the copper(II) centers are reduced. From the pH dependence, it is deduced that the active species of the process is the monohydroxo form of the dinuclear complex. In the second step, the second molecule of catechol is oxidized by the dicopper(I)-dioxygen complex formed upon oxygenation of the reduced complex. In both cases, catechol oxidation is an inner-sphere electron transfer process involving binding of the catechol to the active species. The binary catechol-dicopper(II) complex formed in the first step could be characterized at very low temperature (–90 °C), where substrate oxidation is blocked. On the contrary, the ternary complex of dicopper(I)-O₂-catechol relevant to the second step does not accumulate in solution and could not be characterized, even at low temperature. The investigation of the biphasic kinetics of the catalytic reaction over a range of temperatures allowed the thermodynamic (H° and S°) and activation parameters (H and S) connected with the key steps of the catecholase process to be obtained.     

The catalytic cycle of catechol oxidase

Hybrid density functional theory with the B3LYP functional has been used to investigate the catalytic mechanism of catechol oxidase. Catechol oxidase belongs to a class of enzymes that has a copper dimer with histidine ligands at the active site. Another member of this class is tyrosinase, which has been studied by similar methods previously. An important advantage for the present study compared to the one for tyrosinase is that X-ray crystal structures exist for catechol oxidase. The most critical step in the mechanism for catechol oxidase is where the peroxide O–O bond is cleaved. In the suggested mechanism this cleavage occurs in concert with a proton transfer from the substrate. Shortly after the transition state is passed there is another proton transfer from the substrate, which completes the formation of a water molecule. An important feature of the mechanism, like the one for tyrosinase, is that no proton transfers to or from residues outside the metal complex are needed. The calculated energetics is in reasonable agreement with experiments. Comparisons are made to other similar enzymes studied previously.     

Mitochondrial Dysfunction in Neurodegenerative Diseases Associated with Copper Imbalance

Copper is an essential transition metal ion for the function of key metabolic enzymes, but its uncontrolled redox reactivity is source of reactive oxygen species. Therefore a network of transporters strictly controls the trafficking of copper in living systems. Deficit, excess, or aberrant coordination of copper are conditions that may be detrimental, especially for neuronal cells, which are particularly sensitive to oxidative stress. Indeed, the genetic disturbances of copper homeostasis, Menkes' and Wilson's diseases, are associated with neurodegeneration. Furthermore, copper interacts with the proteins that are the hallmarks of neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, prion diseases, and familial amyotrophic lateral sclerosis. In all cases, copper-mediated oxidative stress is linked to mitochondrial dysfunction, which is a common feature of neurodegeneration. In particular we recently demonstrated that in copper deficiency, mitochondrial function is impaired due to decreased activity of cytochrome c oxidase, leading to production of reactive oxygen species, which in turn triggers mitochondria-mediated apoptotic neurodegeneration.     

Effect of bis(acetato)tetrakis(imidazole) copper(II) in delaying the onset and reducing the mortality rate of strychnine- and thiosemicarbazide—Induced convulsions

The anticonvulsant activity of bis(acetato)tetrakis(imidazole) copper(II), Cu(OAc)₂(Im)₄, was studied in normal mice using chemical convulsions induced by strychnine, thiosemicarbazide, picrotoxin, and pentelenetetrazol. Intraperitoneal administration of Cu(OAc) ₂(Im)₄, 50 mg/kg body mass, has delayed the onset of strychnine (3 mg/kg)-induced convulsion by 204% (p≤0.005) and thiosemicarbazide (20 mg/kg)-induced convulsant by 61% (p≤0.005). The changes in the onset of picrotoxin-(6 mg/kg) and pentelenetetrazol (50 mg/kg)-induced convulsions were not significant. The same dosage of the copper compound was effective in delaying the lethal time and reducing the mortality rate of treated animals. The anticonvulsant activity of Cu(OAc) ₂(Im)₄ complex against strychnine was not related to its constituents because the inorganic form of copper such as copper chloride, copper acetate, and the parent imidazole has no anticonvulsant activity. Other copper(II) complexes like copper(II)aspirinate and bis(acetato)bis(2-methyl imidazole) copper(II) were less effective.     

Oxidative decomposition of vitamin C in drinking water

We have previously shown that vitamin C (ascorbic acid) can initiate hydroxyl radical formation in copper contaminated household drinking water. In the present study, we have examined the stability of vitamin C in copper and bicarbonate containing household drinking water. In drinking water samples, contaminated with copper from the pipes and buffered with bicarbonate, 35% of the added vitamin C was oxidized to dehydroascorbic acid within 15 min. After 3 h incubation at room temperature, 93% of the added (2 mM) ascorbic acid had been oxidized. The dehydroascorbic acid formed was further decomposed to oxalic acid and threonic acid by the hydrogen peroxide generated from the copper (I) autooxidation in the presence of oxygen. A very modest oxidation of vitamin C occurred in Milli-Q water and in household water samples not contaminated by copper ions. Moreover, addition of vitamin C to commercially sold domestic bottled water samples did not result in vitamin C oxidation. Our results demonstrate that ascorbic acid is rapidly oxidized to dehydroascorbic acid and further decomposed to oxalic- and threonic acid in copper contaminated household tap water that is buffered with bicarbonate. The impact of consuming ascorbic acid together with copper and bicarbonate containing drinking water on human health is discussed.     

Calcein as a fluorescent iron chemosensor for the determination of low molecular weight iron in biological fluids

The fluorescence quenching of calcein (CA) is not iron specific and results in a negative calibration curve. In the present study, deferoxamine (DFO), a strong iron chelator, was used to regenerate the fluorescence quenched by iron. Therefore, the differences in fluorescence reading of the same sample with or without addition of DFO are positively and specifically proportional to the amounts of iron. We found that the same iron species but different anions (e.g. ferric sulfate or ferric citrate) differed in CA fluorescence quenching, so did the same anions but different iron (e.g. ferrous or ferric sulfates). Excessive amounts of citrate competed with CA for iron and citrate could be removed by barium precipitation. After optimizing the experimental conditions, the sensitivity of the fluorescent CA assay is 0.02 M of iron, at least 10 times more sensitive than the colorimetric assays. Sera from 6 healthy subjects were tested for low molecular weight (LMW) chelator bound iron in the filtrates of 10 kDa nominal molecular weight limit (NMWL). The LMW iron was marginally detectable in the normal sera. However, increased levels of LMW iron were obtained at higher transferrin (Tf) saturation (1.64–2.54 M range at 80% Tf saturation, 2.77–3.15 M range at 100% Tf saturation and 3.09–3.39 M range at 120% Tf saturation). The application of the assay was further demonstrated in the filtrates of human liver HepG2 and human lung epithelial A549 cells treated with iron or iron-containing dusts.     

Identification of Ion-Selectivity Determinants in Heavy-Metal Transport P<Subscript>1B</Subscript>-type ATPases

P_1B-type ATPases transport a variety of metals (Cd²⁺, Zn²⁺, Pb²⁺, Co²⁺, Cu²⁺, Ag⁺, Cu⁺) across biomembranes. Characteristic sequences CP[C/H/S] in transmembrane fragment H6 were observed in the putative transporting metal site of the founding members of this subfamily (initially named CPx-ATPases). In spite of their importance for metal homeostasis and biotolerance, their mechanisms of ion selectivity are not understood. Studies of better-characterized P_II-type ATPases (Ca-ATPase and Na,K-ATPase) have identified three transmembrane segments that participate in ion binding and transport. Testing the hypothesis that metal specificity is determined by conserved amino acids located in the equivalent transmembrane segments of P_1B-type ATPases (H6, H7, and H8), 234 P_1B-ATPase protein sequences were analyzed. This showed that although H6 contains characteristic CPX or XPC sequences, conserved amino acids in H7 and H8 provide signature sequences that predict the metal selectivity in each of five P_1B-ATPase subgroups identified. These invariant amino acids contain diverse side chains (thiol, hydroxyl, carbonyl, amide, imidazolium) that can participate in transient metal coordination during transport and consequently determine the particular metal selectivity of each enzyme. Each subgroup shares additional structural characteristics such as the presence (or absence) of particular amino-terminal metal-binding domains and the number of putative transmembrane segments. These differences suggest unique functional characteristics for each subgroup in addition to their particular metal specificity.