[Photometric determination of Cu2+ ion using octabromo-tetrakis(4-methylpyridyl)porphine].

Octabromo-tetrakis(4-methylpyridyl)porphine (OBTMPyP), an octabromonated compound with 4 pyrole rings of tetrakis(4-methylpyridyl)porphine, selectively forms a complex with Cu2+ ions at pH 2.0. When 3.6×10(-5) mol/L OBTMPyP was added to the reaction mixture, the calibration curve showed good linearity for Cu2+ ions ranging from 0.01-2.2 µg (addition of 1.0 mL). A good coefficient of variation (Cu2+ ions=1.5 µg (addition of 1.0 mL), n=10, 0.8%) was obtained. The molar absorption coefficient (ε) based on Cu2+ ions was 8.5×10(4) L/mol•cm. This value was 6-fold greater than that determined with a clinical chemical analysis kit using the bathocuproine sulfonic acid method, which is a well-known method for spectrophotometric determination of the Cu2+ ion concentration. A deproteination method was successfully applied in the clinical analysis kit for determination of Cu2+ ion concentrations in control serum I, and the values determined using this method and the bathocuproine sulfonic acid method were almost the same.

[Photometric study on the reaction between 2,3,7,8,12,13,17,18-octabromo- 5,10,15,20-tetrakis-(4-methylpyridyl) porphine and various metal ions].

The chelate forming reaction between 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(4-methylpyridyl)porphine (OBTMPyP) and various metal ions, which belong mainly to 4th period and 7th-12th groups in the periodic table, was examined by the observing the absorption spectra. Because one chemical spicy, H-OBTMPyP, which is one protonated compound at an N atom of pyroll ring among 4 pyroll rings, was observed at pH 9.0, this pH was used to measure the changes of absorption spectra with metal ions. From these changes of absorption spectra of OBTMPyP with metal ions, OBTMPyP were seen to react easily with Cu²âº, Zn²âº, Mn²âº, or Co²âº ion without other additional reagent or heating within 1 min at over 25 °C. On the other hand, OBTMPyP reacted little with Ni²âº, and was not all with Fe³âº (or Fe²âº) reduced by ascorbic acid from Fe³âº) under the same conditions. 5,10,15,20-tetrakis(4-methylpyridyl)porphine (TMPyP) also did not reacted metal ions above these conditions. The λ(max) of each Soret band differed. The stability constants (Ka value) of Cu-, Zn-, Mn- and Co-OBTMPyP was calculated by the change in absorbance of each band, and was 2.6 × 105, 3.6 × 105, 2.7 × 105 and 2.9 × 105 (dm³/mol), respectively. It was revealed that OBTMPyP and metal ions reacted at molar ratio of 1:1, and octabromination of porphine rings improved the reactivity with these ions.

Peroxidase-like catalytic activity of aqueous- and immobilized-Mn(3+)-octabromo-porphyrins on ion-exchange resin supplied as mimetic of horseradish peroxidase.

In order to explore the capability of metal porphyrins as an alternative of horseradish peroxidase (HRP), HRP-like activity of three manganese-porphyrins (Mn-Ps) and three Mn-octabromo-porphyrins (Mn-OBPs) was examined in both aqueous and immobilized states. It was found that Mn(3+)-octabromotetrakis(1-methyl-pyridinium-4yl)porphine (Mn-OBTMPyP) has an activity of at least 90% of HRP in an aqueous solution. Mn-OBTMPyP exhibited a catalytic activity even in the presence of hydrogen peroxide without suicide reaction. In addition, Mn-OBTMPyP was revealed to function as an alternative to HRP in the quantitative determination of serum uric acid. These results are of great interest because they indicate that metal-octabromo-porphyrins possibly include promising candidates of artificial enzyme capable of substituting for HRP.

Effect of carboxyl-group of D-glutamic acid or gamma-carboxy-D-glutamic acid as N-terminal amino acid of (111)in-diethylenetriaminepentaacetic acid-octreotide on accumulation of radioactivity in kidney.

To establish a strategy for developing (111)In-diethylenetriaminepentaacetic acid ((111)In-DTPA)-octreotide, a diagnostic radiopharmaceutical agent for tumors, with reduced non-specific renal radio-accumulation, the compounds having D-glutamic acid (Glu) or gamma-carboxy-D-glutamic acid (carboxy-Glu) as the N-terminal amino acid were examined for in vivo radio-distribution. Compounds carrying Glu and carboxy-Glu containing one and two negative charges, respectively, showed lower renal radio-accumulation than that carrying D-phenylalanine. It was revealed that the introduction of a negative charge reduces the renal radio-accumulation independently from the number of negative charges. The present result can be a clue for the development of (111)In-DTPA-octreotides with reduced the renal radio-accumulation.

Catalytic activity of silica gels bound manganese(III)-porphyrin on oxidative reaction of adrenaline.

To develop a solid catalysis on oxidative reaction of adrenaline (Ad), supports bound metal-tetrakis(4-carboxyphenyl)porphine (M-TCPP) were investigated. Silica gels bound Mn-TCPP were proved to has a superior activity in the oxidation of Ad and be able to serve as a useful oxidase model for Ad.

Peroxidase-like catalytic activity of Mn(3+)-octabromo-tetrakis(4-sulfophenyl)porphine on linoleate hydroperoxide and its analytical application.

To reveal an enzyme-like catalytic activity of metal-octabromo-tetrakis(sulfophenyl)porphines (M-OBPSs), their peroxidease-like catalytic activity on linoleate hydroperoxide (LOOH) were evaluated on the basis of dye-formation in the coloring reaction between N,N-diethylaniline and 4-aminoantipyrine that yields a quinoid-type dye. Among M-OBPSs tested, Mn(3+)-OBPS allowed to produce the largest amount of dye. The optimal conditions of the coloring reaction catalyzed by Mn(3+)-OBPS for the determination of LOOH were determined. A good linear calibration curve was obtained in the concentration range of 0.025-0.4mumole LOOH with good reproducibility (coefficient of variance=1.23%), suggesting that Mn(3+)-OBPS is a good artificial mimesis of the peroxidase for LOOH. In addition, Mn(3+)-OBPS was highly specific for LOOH even in the presence of cumene hydroxyperoxide or hydrogen peroxide. It was revealed that the peroxidase-like activity of Mn(3+)-OBTP is attributable to the redox cycle of Mn(3+)<-->Mn(4+).

Evaluation of the state of active ingredients in pharmaceutical preparations using fourier transform-Raman difference spectra.

To examine the pharmaceutical application of Fourier transform (FT)-Raman spectroscopy, the state of active pharmaceutical ingredients (APIs) in a preparation of several forms was evaluated by investigating the Raman difference spectra between the preparation and excipient. The difference spectra indicated that APIs in alacepril tablets, caffeine sustained-release granules, and quinidine sulfate granules remained unchanged after the manufacturing process. However, the state of sparfloxacin in nanoparticles changed, although it remained unchanged in tablets or powders. These results show that the FT-Raman difference spectrum is expected to be utilized in the field of quality control of crystalline pharmaceutical preparations.

Standard infrared absorption spectrum of betaine and optimal conditions for its measurement.

The infrared absorption (IR) spectrum is often used as a standard reference in identification tests of food additives in Japan. In the case of betaine, many different IR spectra have been reported and, therefore, it is necessary to establish an IR spectrum that is reproducible and reliable enough to be used as a standard for identification. In the present study, suitable conditions to obtain a standard IR spectrum were examined from various viewpoints, including pretreatment, selection of method, and measuring technique. The KBr disk method, which has generally been used to identify betaine, was found to be humidity-dependent, and there was also an interaction between betaine and KBr. A reproducible IR spectrum suitable as a standard could be obtained by drying betaine at 105 degrees C for 3 hours over phosphorus pentoxide, and then measuring the IR spectrum by the liquid paraffin (Nujol) paste method.

HPLC retention behaviors of pi-electron rich compounds on Ni2+- and Cu2+-phthalocyanine derivatives bound to silica gels in polar eluents.

The effect of the central metal of columns packed with silica gels binding Ni(2+)- and Cu(2+)-phthalocyanine derivatives (Ni-and Cu-PCS(D)s) on the retention behavior of poly-aromatic-hydrocarbons (PAHs) thereof in a polar eluent was examined. The retention factors of PAHs on the Ni- and Cu-PCS(D)s in 80% methanol showed a good linear correlation. The Cu-PCS(D) column exhibited the pi-pi interactions for PAHs, while the Ni-PCS(D) column exhibited the pi-d interactions for PAHs in addition to the pi-pi interaction for PAHs. Further, an investigation of the retention behavior of anthracene derivatives having different substituents revealed that the Ni- and Cu-PCS(D) columns could recognize the differences of substituents only in a polar eluent.

Release of vesicular Zn2+ in a rat transient middle cerebral artery occlusion model.

In the brain, Zn(2+) is stored in synaptic vesicles of a subgroup of glutamatergic nerve terminals. Although it has been reported that this Zn(2+) is released upon the excitation of nerves in vitro, there has been little study of the release of Zn(2+) during ischemia in vivo. Here, using brain microdialysis, the release of vesicular Zn(2+) was investigated in vivo. When the vesicular Zn(2+) was released into the synaptic cleft by a depolarizing stimulation achieved by perfusion with Ringer's solution containing high K(+) (100mM KCl), a significant increase in the extracellular concentration of Zn(2+) could be detected by microdialysis. Then, we investigated the release of vesicular Zn(2+) in a rat transient middle cerebral artery occlusion model using microdialysis. Consequently, the extracellular Zn(2+) level in the cortex increased within 15 min of the start of occlusion and reached a peak at 30 min, which was about twice the basal level. After 30 min, it declined with time returning to the basal level 15 min after reperfusion, which was performed after 60 min of occlusion. The results suggest that vesicular Zn(2+) would be released into the synaptic cleft during brain ischemia in vivo.

In vivo measurement of presynaptic Zn2+ release during forebrain ischemia in rats.

Previous studies have suggested that during forebrain ischemia, considerable Zn2+ is released from synaptic vesicles of gultamatergic neuronal terminals and accumulates in hippocampal CA1 pyramidal neurons, leading to delayed neuronal death. However, since a time lag exists between the accumulation of Zn2+ and the occurrence of ischemia and there are conflicting reports about the amount of Zn2+ released, the level of released Zn2+ during ischemia in vivo is still unclear. In this study, we investigated the temporal change of extracellular Zn2+ in the hippocampal CA1 area using microdialysis and the accumulation of Zn2+ in hippocampal CA1 neurons with TSQ staining in rats with a transient forebrain ischemia. The level of extracellular Zn2+ in the CA1 area increased transiently reaching a peak 15 min after occlusion, then decreased with time, returning to the basal level 15 min after reperfusion. In addition, at this peak, the level of extracellular Zn2+ was about twice the basal level. Assessment of the intracellular Zn2+ in hippocampal neurons with TSQ revealed that Zn2+ accumulate at 24 h, but not 0 and 6 h after ischemia. These results suggest that, although the synaptic vesicular Zn2+ is released into the synaptic cleft during ischemia in vivo, the amount of released Zn2+ might not be so excessive, and it does not accumulate in hippocampal CA1 pyramidal neurons immediately after ischemia.

Protective effect of zinc against ischemic neuronal injury in a middle cerebral artery occlusion model.

In this study, we investigated the effect of vesicular zinc on ischemic neuronal injury. In cultured neurons, addition of a low concentration (under 100 microM) of zinc inhibited both glutamate-induced calcium influx and neuronal death. In contrast, a higher concentration (over 150 microM) of zinc decreased neuronal viability, although calcium influx was inhibited. These results indicate that zinc exhibits biphasic effects depending on its concentration. Furthermore, in cultured neurons, co-addition of glutamate and CaEDTA, which binds extra-cellular zinc, increased glutamate-induced calcium influx and aggravated the neurotoxicity of glutamate. In a rat transient middle cerebral artery occlusion (MCAO) model, the infarction volume, which is related to the neurotoxicity of glutamate, increased rapidly on the intracerebral ventricular injection of CaEDTA 30 min prior to occlusion. These results suggest that zinc released from synaptic vesicles may provide a protective effect against ischemic neuronal injury.

HPLC retention behaviors of poly-aromatic-hydrocarbones on Cu(II)-octabromotetrakis(4-carboxyphenyl)porphine derivatives-immobilized aminopropyl silica gels in polar and non-polar eluents.

As one of approaches of developing novel HPLC stationary phases, we prepared Cu-octabromotetrakis(4-carboxyphenyl)porphine derivative-immobilized silica gels (Cu-OBTCPP(D)), and evaluated the availability of the resultant Cu-OBTCPP(D) as a stationary phase for separation of poly-aromatic-hydrocarbons (PAHs) and their related compounds. A Cu-OBTCPP(D) column was revealed to have an ability to separate simple PAHs and be useful as a stationary phase in both polar and non-polar eluents. The retention property of the Cu-OBTCPP(D) column was evaluated in various comparative experiments using commercially available columns. In comparison with 2-(1-pyrenyl)ethyl dimetylsilyl silica gel column (PYE column) regarding the retention behavior for PAHs etc., the Cu-OBTCPP(D) column showed stronger interactions involving pi electron in non-polar eluent than PYE column. In comparison with a pentabromobenzyloxy propylsilyl silica gel column (PBB column) regarding the influence of bromination, the Cu-OBTCPP(D) column was affected differently from the PBB column. In comparison with nitrophenylethyl silica gel column (NPE column) regarding the retention behavior for compounds having a dipole in a non-polar eluent, the Cu-OBTCPP(D) column showed electrostatic interactions such as dipole-dipole interaction equivalent to or larger than the NPE column.

Peroxidase-like catalytic activity of Mn- and Fe-tetrakis(4-carboxyphenyl)porphines bound to aminopropyl-glass bead in oxidative reaction of heterocyclic amines.

Fe- or Mn-tetrakis(4-carboxyphenyl)porphine (Fe- and Mn-TCPP) bound to aminopropyl-glass bead (Fe- and Mn-TCPP(g)s) was examined for the peroxidase (POD)-like function in order to develop a solid catalyst which can exhibit POD-like activity without adsorbing heterocyclic amines (HCAs). Mn-TCPP in aqueous solution had only a slight POD-like catalytic activity on HCAs (IQ and MeIQ). As for Fe-TCPP, it was impossible to examine the POD-like activity since it reacted with hydrogen peroxide in a liquid reaction system. However, both Fe- and Mn-TCPP when immobilized on aminopropyl-glass bead via peptide bond (Fe- and Mn-TCPP(g)s), catalyzed the oxidative reaction of mutagenic HCAs with hydrogen peroxide. The catalytic activity of Fe- and Mn-TCPP(g)s was investigated in more detail using as a substrate IQ and MeIQ which were oxidized more rapidly among the tested HCAs. Consequently, the optimal conditions for the oxidative reaction catalyzed by Fe- and Mn-TCPP(g)s were determined. In addition, ESI-mass and absorption spectra of oxidation products of IQ and MeIQ showed that they are dimers. Thus, it was demonstrated that a solid catalyst with POD-like activity can be obtained by immobilizing Fe- and Mn-TCPPs on aminopropyl-glass beads.

Effect of octabromination of a tetrakis(4-carboxyphenyl)porphine derivative bound to silica gels on HPLC retention behaviors of polyaromatic hydrocarbons.

The effect of bromination of Cu-porphyrin-derivative-immobilized silica gels (Cu-TCPP(D)) was examined by comparing the retention behaviors of polyaromatic hydrocarbons (PAHs) on Cu-TCPP(D) and Cu-octabromotetrakis(4-carboxyphenyl)porphine-derivative-immobilized silica gels (Cu-OBTCPP(D)) columns. It was revealed that bromination affects strongly the pi-pi electron interactions caused from hydration energy in a polar eluent (80% methanol) possibly as a result of a destruction of planar structure of porphine-ring by bromination. It was also revealed that bromination enhances pi-d interactions as well as the pi-pi electron interactions in a broad sense (e.g., dispersion forces) in a non-polar eluent (n-hexane). However, the bromination did not exert much influence on electrostatic interactions caused from polarization of mono-halogenated benzenes.

Peroxidase-like catalytic activity of anion-exchange resins modified with metal-porphyrins in oxidative reaction of hetrocyclic amines with hydrogen peroxide.

To elucidate the peroxidase (POD)-like catalytic activity of anion-exchange resins modified with metal-tetrakis(sulfophenyl)porphine (M-TSPP(r)s), an oxidative reaction of seven mutagenic heterocyclic amines (HCAs) with hydrogen peroxide, which reaction is catalyzed by horse radish POD, was investigated in the presence of M-TSPP(r)s. Among six M-TSPP(r)s tested, Mn- and Fe-TSPP(r)s were found to have a relatively strong POD-like activity for HCAs, in particular for a typical HCA, 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ). The optimal condition for the POD-like activity was selected using Fe- and Mn-TSPP(r)s. For evaluation of an oxidation product of IQ produced in the presence of Fe-TSPP(r), the absorption, NMR and FAB-mass spectra thereof were compared with those of an oxidation product of IQ produced by horse radish POD or a chemical oxidizing agent, sodium hypochlorite. When Fe-TSPP(r) was present as a catalysts, IQ was converted into the dimmer (hydorazone type) which has no mutagenic activity in umu-test. It was revealed that Fe- and Mn-TSPP(r)s exhibit a POD-like catalytic activity in oxidative reaction of HCAs with hydrogen peroxide.

Quantitative determination method for trace amount of penicillin contaminants in commercially available drug product by HPLC coupled with tandem mass spectrometry.

A quantitative determination method for trace amount of penicillin contaminants in an active pharmaceutical ingredient (API) has been developed. Selective extraction of penicillin contaminants from the matrix containing API and specific separation among penicillin contaminants were achieved through an on-line column switching technique with gradient elution, followed by tandem mass spectrometric determination. Validation was conducted on the developed method in terms of specificity, linearity, accuracy, precision, and detection limit, and appeared reasonable. The detection limit was estimated as 0.03 ng/ml or lower of the concentration of penicillin contaminants in the preparation, corresponding to 4 parts par billion (ppb) against the API. This fulfilled the regulatory requirement by the authorities.

Inhibitory effects of water-soluble cationic manganese porphyrins on peroxynitrite-induced SOS response in Salmonella typhimurium TA4107/pSK1002.

We have investigated the protective effects of water-soluble cationic Mn(III) porphyrins against peroxynitrite (ONOO-)-induced DNA damage in the cells of Salmonella typhimurium TA4107/pSK1002 and lipid peroxidation of red blood cell membranes. Mn(III) tetrakis (N-methylpyridinium-4-yl) porphine (TMPyP) and the brominated form, Mn(III) octabromo-tetrakis (N-methylpyridinium-4-yl) porphine (OBTMPyP) effectively reduced the damage and peroxidation induced by N-morpholino sydnonimine (SIN-1), which gradually generates ONOO- from O2*- and *NO produced through hydrolysis. Mn(III)OBTMPyP became 10-fold more active than the non-brominated form. In the presence of authentic ONOO-, the Mn(III) porphyrins were ineffective against damage and strongly enhanced lipid peroxidation, while the coexistence of ascorbic acid inhibited peroxidation. Using a diode array spectrophotometry, the reactions of Mn(III)TMPyP with authentic ONOO- and SIN-1 were measured. Mn(III)TMPyP is known to be catalytic for ONOO- decomposition in the presence of antioxidants. OxoMn(IV)TMPyP with SIN-1 was rapidly reduced back to Mn(III) without adding any oxidants. Further, in the SIN-1 system, the concentration of NO2- and NO3- were colorimetrically determined by Griess reaction based on the two-step diazotization. NO2- increased by addition of Mn(III) porphyrin and the ratio of NO2- to NO3- was 4-7 times higher than that (1.05) of SIN-1 alone. This result suggests that O2*- from SIN-1 acts as a reductant and *NO cogenerated is oxidized to NO2-, a primarily decomposition product of *NO. Under the pathological conditions where biological antioxidants are depleted and ONOO- and O2*- are extensively generated, the Mn(III) porphyrins will effectively cycle ONOO- decomposition using O2*-.

Coloration of phenothiazines with metal-containing drugs.

We studied the color change of phenothiazines and metal-containing drugs after compound formation, followed by use of FT-Raman spectrocopy to observe any structural changes in them. When 6 phenothiazines (thioridazine hydrochloride, prochlorperazine maleate, levomepromazine maleate, chlorpromazine phenolphthalinate, fluphenazine maleate and perphenazine fendiate) formed compounds with natural aluminum silicate, the color change was accompanied by a shift of FT-Raman signals. These changes could be attributed to the structural changes of phenothiazines. This present observation can be then used in advance to avoid coloration of phenothiazines during preparative procedures with metal-containing drugs such as antacids.

Effect of carboxylation of N-terminal phenylalanine of (111)in-DTPA (diethylenetriaminepentaacetic acid)-octreotide on accumulation of radioactivity in kidney.

For purpose of reducing renal accumulation of radioactivity of a known radiopharmaceutical agent, i.e., (111)In-DTPA (diethylenetriaminepentaacetic acid)-D-Phe(1)-octreotide, a derivative in which p-carboxy-L-phenylalanine is substituted for D-Phe(1) was synthesized. Biodistribution study of the resultant compound having carboxy-substituted L-Phe(1) revealed that the renal accumulation was significantly lower than that of control compound having unsubstituted L-Phe(1), demonstrating that the presence of negative charge on the N-terminal amino acid of octreotide is effective to reduce the renal accumulation. This effect can be attributed to the reduction of lipophilicity and also the repulsive force arisen from the negative charge of renal brush border membrane.