Complex-formation and redox reactions of bilirubin and biliverdin with zinc(II), cadmium(II) and copper(II) ions

Transition metal complexes of bilirubin and biliverdin were studied spectrophotometrically, in DMSO and in a boric acid-NaOH buffer mixture at pH 10.5. In the zinc(II) and cadmium(II)- bilirubin systems, 2:1 complexes are formed. Both in aqueous and in DMSO medium, the copper(II) ion oxidizes bilirubin to biliverdin. With all three metal ions, biliverdin forms 1:1 complexes, the stabilities of which are higher than those of the corresponding bilirubin complexes. Accordingly, these metal ions accelerate the oxidative transformations of bilirubin.     

Structural study of few p-phenylenediacetate complexes of Mn, Cu, Zn and Cd

Different metal carboxylate complexes are prepared by reactions of manganese (+2) acetate, copper (+2) acetate, zinc (+2) acetate and cadmium (+2) acetate with p-phenylenediacetic acid under ambient condition with or without a nitrogen donor ligands and each of them are characterized by conventional spectroscopic techniques along with crystallography.     

Synthesis and characterization of pyridine N-oxide complexes of manganese, copper and zinc

A series of metal carboxylates containing pyridine N-oxide are prepared via one pot synthesis and solid phase synthesis. The structural variations from metal to metal are observed. In the case of reactions of manganese(II) acetate with pyridine N-oxide in the presence of aromatic carboxylic acids, polymeric complexes with bridging aromatic carboxylate as well as bridging pyridine N-oxide are observed. Whereas, the reaction of copper(II) acetate with pyridine N-oxide in the presence of an aromatic carboxylic acid led to mononuclear or binuclear paddle wheel carboxylate complexes with monodentate pyridine N-oxide. Co-crystal of two neutral complexes having composition [Cu₂(OBz)₄(MeOH)₂][Cu₂(OBz)₄(pyO)₂] (where OBz = benzoate, pyO = pyridine N-oxide) each neutral parts have paddle wheel structure. Solid phase reaction of zinc chloride with sodium benzoate prepared in situ and pyridine N-oxide leads to a tetra-nuclear zinc complex.     

Effect of zinc on the Stokes' radius of metallothionein

Apothionein was prepared from rat liver metallothionein-II. To this, zinc, as zinc acetate, was re-added in a stepwise fashion to yield a series of complexes with ratios of metal to protein varying from 1:1 to 7:1. The Stokes' radius of the apoprotein and each zinc-metallothionein complex was then determined by gel filtration. A plot of atoms Zn/mol metallothionein versus Stokes' radius yielded a sigmoid curve, with values for Stokes' radius ranging from 20.8 A for the apoprotein 15.4 A for the fully saturated zinc complex.     

Steric effects in controlling co-ordination environment in zinc 2-nitrobenzoate complexes

A dinuclear zinc complex having exclusive bis(unidentate) carboxylato co-ordination mode is synthesized through two-step reactions, involving solid phase reaction followed by solution reaction. The formation of such dinuclear species is governed by the ligand under consideration. For example, 2-nitrobenzoate zinc complex having 1,10-phenanthroline is mononuclear whereas a similar complex with 8-aminoquinoline is dinuclear having bis(unidentate) carboxylato co-ordination mode. The reasons for the formation such complex is explained on the basis of the distance of separation of uncoordinated oxygen atoms of the carboxylate oxygen of mononuclear and dinuclear complexes. Zinc 2-nitrobenzoate complexes having 2,2′-bipyridine is mononuclear but with 2-aminopyrimidine has a dinuclear paddle wheel structure. Solution phase reaction of 2-nitrobenzoic acid with zinc(II)acetate in the presence of 8-aminoquinoline gives bis-(8-aminoquinoline)zinc(II) 2-nitrobenzoate.     

New Zn complexes based on 1,2,4-triazoles: Synthesis, structure and luminescence

The reaction of 3-(pyridin-2-yl)-5-(2-aminophenyl)-1H-1,2,4-triazole and salicylaldehyde or benzaldehyde leads a new type of 1,2,4-triazole’s derivats. Reactions of these ligands with zinc acetate in ethanol lead to the formation of two new complexes with azomethine or dihydrotriazaindolizine form of ligands. The structures of these complexes were investigated by X-ray analysis. The complexes showed strong green-blue luminescence in solid state.     

Syntheses and structures of M(L)(X)BPh4 complexes {M=Co(II), Zn(II); L=cis-1,3,5-tris[3-(2-furyl)prop-2-enylideneamino]cyclohexane, X=OAc, NO3}: structural models of the active site of carbonic anhydrase

The metal coordination geometries in the structures of the zinc(II) and cobalt(II) complexes of the ligand cis-1,3,5-tris[3-(2-furyl)prop-2-enylideneamino]cyclohexane (fr-protach) and with the anions nitrate and acetate are structural models for the active site of carbonic anhydrase. The acetate structures show a striking structural correlation with the metal coordination environments in the known bicarbonate forms of the enzyme. Such structures provide a basis for understanding the marked effect of different metal substitution on the catalytic rate of the enzyme.     

Gold complexes inhibit mitochondrial thioredoxin reductase: consequences on mitochondrial functions

The effects of gold(I) complexes (auranofin, triethylphosphine gold and aurothiomalate), gold(III) complexes ([Au(2,2'-diethylendiamine)Cl]Cl(2), [(Au(2-(1,1-dimethylbenzyl)-pyridine) (CH(3)COO)(2)], [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine)(OH)](PF(6)), [Au(bipy(dmb)-H)(2,6-xylidine)](PF(6))), metal ions (zinc and cadmium acetate) and metal complexes (cisplatin, zinc pyrithione and tributyltin) on mitochondrial thioredoxin reductase and mitochondrial functions have been examined. Both gold(I) and gold(III) complexes are extremely efficient inhibitors of thioredoxin reductase showing IC(50) ranging from 0.020 to 1.42 microM while metal ions and complexes not containing gold are less effective, exhibiting IC(50) going from 11.8 to 76.0 microM. At variance with thioredoxin reductase, auranofin is completely ineffective in inhibiting glutathione peroxidase and glutathione reductase, while gold(III) compounds show some effect on glutathione peroxidase. The mitochondrial respiratory chain is scarcely affected by gold compounds while the other metal complexes and metal ions, in particular zinc ion and zinc pyrithione, show a more marked inhibitory effect that is reflected on a rapid induction of membrane potential decrease that precedes swelling. Therefore, differently from gold compounds, the various metal ions and metal complexes exert their effect on different targets indicating a lower specificity. It is concluded that gold compounds are highly specific inhibitors of mitochondrial thioredoxin reductase and this action influences other functions such as membrane permeability properties. Metal ions and metal complexes markedly inhibit the activity of thioredoxin reductase although to an extent lower than that of gold compounds. They also inhibit mitochondrial respiration, decrease membrane potential and, finally, induce swelling.     

An estimation of the equivalent solution dielectric constant in the active-site cavity of metalloenzymes. Dependence of carboxylate-metal-ion complex stabilities on the polarity of mixed aqueous/organic solvents

The stability constants of the 1:1 complexes between Cu2+ and Zn2+ with formate, acetate and several phenylalkanecarboxylates, i.e. C6H5-(CH2)n-COO- with n = 0 to 5, are summarized for water, 50% aqueous ethanol and 50% aqueous dioxane (I = 0.1 M; 25 degrees C): Complex stability depends upon carboxylate group basicity. The influence of varying amounts of ethanol or dioxane (up to 90%) on the stability of the Cu2+ and Zn2+ (M2+) complexes with formate and acetate (CA) was measured by potentiometric pH titrations. The values for pKHH(CA) and log KMM(CA) increase, as expected, with increasing amounts of the organic solvents, i.e. with decreasing solvent polarity. The changes in the equilibrium constants are also evaluated with regard to the mole fractions of the organic solvents and the corresponding dielectric constants. These results may be used to estimate for low dielectric cavities in proteins the equivalent solution dielectric constant on the basis of enhanced carboxylate basicity or metal ion binding capability (method 1). Furthermore, the measured stability constants are used for comparisons of the coordination tendency of carboxylate ligands towards zinc(II)-metalloenzymes (method 2); in this way the equivalent solution dielectric constants in the active-site cavities of bovine carbonic anhydrase and carboxypeptidase A are estimated: the values are of the order of 35 and 70, respectively. This method seems to be generally applicable to metalloproteins.     

Use of cyclodextrins as a cosmetic delivery system for fragrance materials: Linalool and benzyl acetate

The aim of this study was to increase the stability and water solubility of fragrance materials, to provide controlled release of these compounds, and to convert these substances from liquid to powder form by preparing their inclusion complexes with cyclodextrins (CDs). For this purpose, linalool and benzyl acetate were chosen as the fragrance materials. The use of beta-cyclodextrin (beta CD) and 2-hydroxypropyl-beta-cyclodextrin (2-HP beta CD) for increasing the solubility of these 2 fragrance materials was studied. Linalool and benzyl acetate gave a B-type diagram with beta CD, whereas they gave an A(L)-type diagram with 2-HP beta CD. Therefore, complexes of fragrance materials with 2-HP beta CD at 1:1 and 1:2 molar ratios (guest:host) were prepared. The formation of inclusion complexes was confirmed using proton nuclear magnetic resonance ((1)H-NMR) spectroscopy and circular dichroism spectroscopy. The results of the solubility studies showed that preparing the inclusion complex with 2-HP beta CD at a 1:1 molar ratio increased the solubility of linalool 5.9-fold and that of benzyl acetate 4.2-fold, whereas the complexes at a 1:2 molar ratio increased the solubility 6.4- and 4.5-fold for linalool and benzyl acetate, respectively. The stability and in vitro release studies were performed on the gel formulations prepared using uncomplexed fragrance materials or inclusion complexes of fragrance materials at a 1:1 molar ratio. It was observed that the volatility of both fragrance materials was decreased by preparing the inclusion complexes with 2-HP beta CD. Also, in vitro release data indicated that controlled release of fragrances could be possible if inclusion complexes were prepared.     

Application of a rapid and efficient h.p.l.c. method to measure bilirubin and its conjugates from native bile and in model bile systems. Potential use as a tool for kinetic reactions and as an aid in diagnosis of hepatobiliary disease.

We have developed an extremely rapid and efficient reverse-phase h.p.l.c. method for the measurement of bilirubin and its conjugates in human bile and in model bile systems. Our method involves the use of a Perkin-Elmer 3 mu C18 column and a methanol/sodium acetate/aq. ammonium acetate buffer system. Three isomers of bilirubin diglucuronide (BDG), two isomers of bilirubin monoglucuronide (BMG), three isomers of unconjugated bilirubin (UCB) and minor conjugates containing glucose and xylose were separated in 12 min. Initial quantification of BDG and BMG was based on the use of the ethyl anthranilate azo derivative of bilirubin (AZO UCB); however, the standard curves for BDG, BMG and UCB were similar enough to permit quantification to be later based on the UCB standard curve only, thereby simplifying the quantification process. Routine direct injection of 6 or 10 microliter of crude undiluted or diluted (1:1) bile sample was sufficient for analysis. The method was helpful in diagnosing biliary-tract obstruction in a newborn and a partial deficiency state of bilirubin conjugation (Crigler-Najjar syndrome) in a 10-year-old male. When the method was applied to biles of patients both with and without gallstones, levels of UCB were less than 2% of total pigment, consistent with previous reports. Because of its speed and efficiency, this method has the potential for a broad range of applications including enzymic, kinetic and bile sample analyses.     

Spectroscopic properties of bilirubin-human serum albumin complexes: a stoichiometric analysis

Light absorption spectra, fluorescence of bound bilirubin, fluorescence of albumin as quenched by bilirubin, and circular dichroism spectra have been studied in mixtures of bilirubin and defatted human serum albumin in variable proportions at 25 degrees C and at pH 7.4, 8.2, and 9.0. Corresponding spectral data have been calculated for the stoichiometric bilirubin-albumin complexes, 1:1, 2:1, and 3:1. Light absorption spectra as well as the bound bilirubin fluorescence indicate that all three bound bilirubin dianions are internalized. These data were obtained by curve fitting to least sum of squared deviations. In addition to the best fit we obtained 30 acceptable curves, located within an F contour, thus producing a rough estimate of the variation of the resulting spectral data.     

Model complexes of the active site of galactose oxidase. Effects of the metal ion binding sites

Model compounds of the active site of galactose oxidase have been developed by using new cofactor model ligands, L1H (2-methylthio-4-tert-butyl-6-[{bis(pyridin-2-ylmethyl)amino}methyl]phenol) and L2H (2-methylthio-4-tert-butyl-6-[{bis(6-methylpyridin-2-ylmethyl)amino}methyl]phenol). Treatment of the ligands with copper(II) and zinc(II) perchlorate in the presence of triethylamine followed by anion exchange reaction with NaPF₆ or NaBPh₄ provided the corresponding copper(II) and zinc(II) complexes, the crystal structures of which have been determined by X-ray crystallographic analysis. All the copper(II) and zinc(II) complexes have been isolated as a dimeric form in which the phenolate oxygen of each ligand acts as the bridging ligand to form a rhombic M₂(OAr)₂ core (M=Cu or Zn). The dimeric complexes can be converted into the corresponding monomer complexes by the treatment with exogenous ligand such as acetate ion. The redox potential and the spectroscopic features of the monomer complexes have also been examined. Furthermore, the copper(II)- and zinc(II)-complexes of the phenoxyl radical species of the ligands have been generated in situ by the oxidation of the phenolate complexes with (NH₄)₂[Ce^IV(NO₃)₆] (CAN) in CH₃CN, and their spectroscopic features have been explored. The structures and physicochemical properties of the phenolate and phenoxyl radical complexes of L1 and L2 have been compared to those of the previously reported copper(II) and zinc(II) complexes of L3 (2-methylthio-4-tert-butyl-6-[{bis(2-pyridin-2-ylethyl)amino}methyl]phenol) in order to get insights into the interaction between the metal ions and the organic cofactor moiety.     

Effect of Bilirubin on the Membrane Potential of Rat Brain Synaptosomes

The effect of the neurotoxic pigment bilirubin on the membrane potential of rat brain synaptosomes was studied by using the tetraphenylphosphonium ion (TTP+) technique. Bilirubin induces a rapid depolarization of synaptosomes, as reflected by an efflux of previously accumulated [3H]TTP+. This phenomenon persisted when the membrane potential across either the plasma membrane of the synaptosome or the inner membrane of the entrapped mitochondria was selectively depressed, thus indicating that both components of the synaptosomal membrane potential were affected by bilirubin. Bovine serum albumin, used at a albumin/bilirubin molar ratio of 1:1, had the capacity to completely prevent and reverse the effect of bilirubin. This fact demonstrates that the bilirubin-induced TPP+ release from synaptosomes is a reversible process that requires the presence of bilirubin interacting with the synaptosomal membranes. These results, together with the inhibition by bilirubin of [3H]TPP+ and [2-14C]acetate uptake by synaptosomal plasma membrane vesicles isolated from rat brain, suggest that bilirubin depresses the membrane potential across the synaptosomal plasma membrane by a mechanism involving alterations in ion permeability. This effect could be of relevance in the pathogenesis of bilirubin encephalopathy.     

Low molecular weight copper-binding ligands in human bile

The aim of this study was to detect and identify major low molecular weight (less than 10,000) copper-binding ligands in human bile. Modified gel chromatography was used as the method of ligand detection because it ensures the detection of labile as well as inert metal-ligand complexes. Conjugated bilirubin, peptides, and amino acids, primarily glycine, were isolated as the major ligands. In contrast to the other copper-binding ligands, the peptides were poor zinc binders, suggesting the possibility that they may confer necessary specificity to trace metal elimination.     

Separation and sensitive determination of i-urobilin and 1-stercobilin by high-performance liquid chromatography with fluorimetric detection

i-Urobilin and 1-stercobilin were separated by high-performance liquid chromatography on a reversed-phase octadecylsilane-bonded column and detected fluorimetrically through formation of phosphor with zinc ions in the eluent. The separation and the intensity of the fluorescence response were affected by concentrations of zinc acetate and sodium borate buffer, pH and methanol content in the eluent. The optimal eluent used consisted of 0.1% zinc acetate in 75 mM boric acid buffer (pH 6.0)—methanol (25:75). The detection limit was 0.2 μg/l for both i-urobilin and 1-stercobilin (signal-to-noise ratio 2), which makes the method 250–2500 times more sensitive than conventional methods.     

Bilirubin production and conjugation from newly formed heme in isolated rat hepatocytes.

1. Heme synthesis from delta-aminolevulinic acid (delta-ALA) in freshly isolated rat hepatocytes was maximal at 100 microM with a rate of approx. 7 nmol being synthesized per g wet weight cells. 2. Approximately 8% of synthesized heme was converted to bilirubin and 50% of the newly synthesized bilirubin was conjugated. 3. The ratio of di to monoconjugate was approx. 2.5. Incorporation of delta-ALA into bilirubin was increased by additional delta-ALA, heme and was also doubled in cells isolated from animals treated with CoCl2. 4. Bilirubin formation was inhibited approx. 90% by in vitro treatment with heme oxygenase inhibitors zinc and tin protoporphyrin.     

Investigation of excretion and absorption of different zinc salts in puppies.

The effects of dietary zinc on zinc absorption and excretion were evaluated with six 12-week-old beagles. The dogs were fed a commercial dry food later supplemented with 2 and 4mg/kg of body weight per day of either zinc sulphate, zinc acetate or zinc oxide. The concentrations of zinc in the urine and faeces of all treatment groups were increased depending on the type of zinc salts and the dosage in the diet (P < 0.05). The apparent absorption of zinc salts ranged from 0.20 to 0.36. The zinc salt supplements at both dosages did not affect the digestibility of crude protein, fat and fibre. The zinc concentration in plasma varied with the type of zinc salt and with the dosage. The mean concentrations ranged from 61+/- 1.46 microg to 73 +/- 1.57 microg/dl in the unsupplemented groups, whereas it was 115 +/- 2.33 microg/dl in the group supplemented with zinc acetate at the highest dosage.     

Hepatic membranolytic stability alteration by metalloporphins in rats.

The mothers of experimental neonates were administered excess bilirubin for a month, and the neonates were suffering from hyperbilirubinemia. The studies were conducted on the effect of excess bilirubin and metalloporphyrins on plasma membrane and mitochondrial membrane. We have isolated, separated, and estimated phospholipids, and also assayed the activity of phospholipase A2 from whole liver and mitochondrial and microsomal fractions. Excess of bilirubin administration decreased the total phospholipid level and inhibited the phospholipase A2 activity. Cr-PP (chromium protoporphyrin) induces the phospholipase A2 activity which is inhibited by simultaneous bilirubin administration. However, Zn-PP (zinc protoporphyrin) and Mn-PP (manganese protoporphyrin) showed a reverse pattern.     

Estimation of apparent quadrupolar coupling constants for complexes of magnesium ions with mono- and dicarboxylic acid ligands. Applications to magnesium ion: protein interactions

25Mg+2 ion NMR studies of complexes of magnesium ions with acetate and malonate ligands have yielded apparent quadrupolar coupling constants, chi, of approximately 1.5 MHz. The aquo magnesium ion yields a smaller chi value of 0.12 MHz, consistent with its expected higher symmetry. chi values for magnesium ion: acetate and magnesium ion: malonate complexes are utilized to calculate observed linewidths for magnesium ion: bovine prothrombin fragment 1 and magnesium ion: human Factor XII interactions. These calculated values are compared with observed values and implications of the agreement are discussed.