Chiyo Matsubara

Oxo[5, 10, 15, 20-tetra(4-pyridyl)porphyrinato]titanium(IV): an ultra-high sensitivity spectrophotometric reagent for hydrogen peroxide

The water-soluble titanium(IV)–porphyrin complex, oxo[5, 10, 15, 20-tetra(4-pyridyl)porphyrinato]titanium(IV)[TiO(tpyH4)4+], was found to enhance the spectrophotometric determination of trace amounts of hydrogen peroxide. A 0.05 mol dm–3 hydrochloric acid solution containing TiO(tpypH4)4+ was used (the Ti—TPyP reagent), the absorbance of which decreased at 432 nm as hydrogen peroxide was added. This was due to the consumption of the TiO(tpypH4)4+ complex following the formation of peroxo[5, 10, 15, 20-tetra(4-pyridyl)porphyrinato]titanium(IV). The decrease in absorbance at 432 nm (ΔA432) was proportional to the concentration of hydrogen peroxide, from 1.0 × 10–8 to 2.8 × 10–6 mol dm–3, in the sample solution (25 pmol–7.0 nmol per assay). The reaction was accelerated by hydrogen ions; the presence of 1.6 mol dm–3 perchloric acid was found to promote complexation to the greatest extent. A ΔA432 of 1.9 × 105 was found for 1 mol dm–3 hydrogen peroxide. A measurement precision of 1.2% for 1.0 × 10–6 mol dm–3 hydrogen peroxide (n= 8) was obtained. The reagent can be used for the determination of hydrogen peroxide in water samples such as tap water and rainwater over the range from 1.05 × 10–7 to 3.34 × 10–5 mol dm–3.

A spectrophotometric method for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-coenzyme A oxidase

A mixture of Ti(IV) and 4-(2-pyridylazo)resorcinol was found to be useful in the spectrophotometric determination of trace amounts of hydrogen peroxide. The absorbance at 508 nm was proportional to the concentration of hydrogen peroxide added. The reagent was successfully applied to the assay of free fatty acid in serum through the combined use of acyl-CoA synthetase and acyl-CoA oxidase. The latter enzyme produces H2O2. As a result, hydrogen peroxide was produced through the enzymatic oxidation of free fatty acid. It was possible to determine free fatty acid in 50 microliters of serum at concentrations ranging from 0.02 to 1.5 mM. The coefficient of variation was less than 3% at concentrations ranging from 0.1 to 1.5 mM. In the present method, there is the advantage of minimal influence from reducible substances as well as greater simplicity and accuracy.

Polymer-mediated extraction of 8-quinolinolato metal chelates for the determination of metal ions in water with graphite furnace atomic absorption spectrometry.

Water-insoluble 8-quinolinolato metal chelates were formed and were stably solubilized in the aqueous solution of a water-soluble polymer, poly (N-isopropylacrylamide)(PNIPAAm), at room temperature. When the solution was heated at 50 degrees C, PNIPAAm precipitated and then formed a gum-like aggregate (polymer phase) having a very small volume. Accompanying the polymer precipitation, hydrophobic 8-quinolinolato chelates with cobalt(II), iron(III), nickel(II), and copper(II) ions were efficiently incorporated into the polymer phase. At 0.5% (w/v) of PNIPAAm and 8.0 mM of 8-quinolinol, the recoveries in the incorporation of four metal chelates were quantitative. The fluorescence spectra of a probe suggests that the hydrated polymer in the aqueous solution provides hydrophobic portions which can incorporate hydrophobic metal chelates. The polymer phase was easily taken out from the solution and was dissolved with a small amount of acetonitrile. The resulting solution could be directly introduced into a graphite furnace of atomic absorption spectrometry. The signal intensities for the absorbance of cobalt after concentrating the chelate were 100-fold greater than those before the concentration.

Different role of serum components and cytokines on alveolar macrophage activation by soluble fungal (1→3)-β-d-glucan

In this study, we investigated the mechanism of alveolar macrophage activation by systemic administration of SSG, a soluble highly branched (1-->3)-beta-D-glucan obtained from a fungus Sclerotinia sclerotiorum IFO 9395. Multiple i.v. administration (10 mg/kg; once daily for 10 consecutive days) of SSG enhanced some functions of alveolar macrophages, such as lysosomal enzyme activity and nitric oxide secretion, on day 1 after the last administration, and it also elevated the concentrations of serum protein, interferon gamma and SSG in bronchoalveolar lavage fluid on the same day. On the in vitro assay system, stimulation by SSG alone (500 microg/ml) slightly augmented the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide production of cells. Stimulation by serum (1 or 10% mouse serum) or serum components, such as fibronectin (25 microg/ml) and albumin (500 microg/ml), alone strongly augmented only the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide secretion from cells, and no synergism or additive-like effect was observed between serum components and SSG. In contrast, stimulation by crude lymphokine (5%) or recombinant murine interferon gamma (100 U/ml) alone did not induce augmentation of lysosomal enzyme activity and nitric oxide production of alveolar macrophages in vitro, but when cells were incubated together with crude lymphokine or recombinant murine interferon gamma and SSG (500 microg/ml), a significant combined effect was observed on both functions of alveolar macrophages. In addition, pretreatment of crude lymphokine or recombinant murine interferon gamma enhanced the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface in vitro though pretreatment by serum components had no effect. Based on these findings, the enhancement of alveolar macrophage functions by systemic administration of SSG appears to be mediated, at least in part, by both the simple effect of serum components including fibronectin and albumin leaked from pulmonary peripheral blood into the alveoli and the synergistic effect between lymphokines released from activated pulmonary T cells and SSG itself entering the alveoli after SSG injection via the priming effect of lymphokines which enhances the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface.

Cytotoxicological aspects of organic arsenic compounds contained in marine products using the mammalian cell culture technique

Arsenobetaine, arsenocholine, trimethylarsine oxide and tetramethylarsonium iodide, which are contained in marine fishery products, were examined for their potencies on cell growth inhibition, chromosomal aberration and sister chromatid exchange (SCE). Arsenobetaine, the major water-soluble organic arsenic compound in marine animals, exhibited very low cytotoxicity towards mammalian cells. This compound showed no cell growth inhibition at a concentration of 10 mg cm -3 and the cytotoxicity was lower than 1/14 000th of that of sodium arsenite and 1/1600th of that of sodium arsenate towards BALB/c 3T3 cells. The chromosomal aberrations caused by arsenobetaine at a concentration of 10 mg cm -3 consisted mainly of chromatid gaps and chromatid breaks, but in this concentration chromosomal breakage owing to its osmotic pressure is likely to be considerable. No SCE was observed at a concentration of 1 mg cm -3 . Arsenocholine and trimethylarsine oxide also showed no cell growth inhibited at a concentration of 10 mg cm -3 . However, tetramethylarsonium iodide inhibition the growth of BALBIc 3T3 at a concentration of 8 mg cm -3 . These compounds exhibited a low ability to induce chromosomal aberrations at a concentration range of 2-10 mg cm -3 and no SCE was observed at a concentration of 1.0 mg cm -3 . These results suggested that the major and minor organic arsenic compounds contained in marine fishery products are much less cytotoxic inorganic arsenic, methylarsonic acid and dimethylarsinic acid.

Rapid determination of trace amounts of phosphate and arsenate in water by spectrophotometric detection of their heteropoly acid-malachite green aggregates following pre-concentration by membrane filtration

A simple and rapid method for the determination of trace amounts of phosphate and arsenate in water is proposed. Molybdophosphate-and molybdoarsenate-Malachite Green aggregates, formed by reaction of a reagent consisting of a mixed solution of ammonium molybdate and Malachite Green, were selectively collected on a nitrocellulose membrane filter (pore size 3 µm) and dissolved in methylcellosolve together with the membrane filter. The absorbance (λ= 627 nm), denoted A(P + As), was proportional to the sum of the concentrations of phosphate and arsenate with a molar absorptivity of 2.7 × 105 l mol–1 cm–1. Thiosulphate, a reducing agent for arsenate, was added to water samples, and the absorbance of the solution, A(P), was measured as described above. The absorbance A(P) corresponds to the concentration of phosphate alone. The difference, A(P + As)–A(P), then corresponds to the arsenate concentration. The proposed method makes it possible to determine phosphate and arsenate at levels ranging from 0.3 to 150 p.p.b.

Immunotoxicity of organic arsenic compounds in marine animals

In the present study, we demonstrated for the first time the immunotoxic effects of organic arsenic compounds in marine animals, namely arsenocholine [AsCho; trimethyl(2-hydroxyethyl)arsonium cation], arsenobetaine [AsBe; the trimethyl(carboxymethyl)arsonium zwitterion] and the tetramethylarsonium ion (TetMA), to murine principal immune effector cells (macrophages and lymphocytes), comparing them with the effects of inorganic arsenicals in vitro. Inorganic arsenicals (arsenite and arsenate) showed strong cytotoxicity to both macrophages and lymphocytes. The concentration of arsenite that reduced the number of surviving cells to 50% of that in untreated controls (IC 50 ) was 3-5 μmol dm -3 , and the cytotoxicity of arsenate (IC 50 =100 μ-1 m mol dm -3 ) was lower than that of arsenite. Compared with these findings, trimethylarsenic compounds in marine animals, AsCho and AsBe, were less toxic even at a concentration over 10 mmol dm -3 to both macrophages and lymphocytes; however, TetMA had weak, but significant, cytotoxicity to these cells (IC 50 was about 6 mmol dm -3 ).

Micelle-mediated extraction for concentrating hydrophobic organic compounds.

An extraction method based on polymer-induced phase separation of aqueous micellar solutions of octyl-beta-D-thioglucoside (OTG) was assessed for concentrating hydrophobic analytes. Various hydrophobic compounds such as polycyclicaromatic hydrocarbons, alkylbenzenes, alkylphenols, chlorobenzenes, chlorophenols, phthalic esters, pesticides, and steroid hormones could be efficiently concentrated into a small volume of surfactant-rich phase, while hydrophilic matrix components remained in the bulk aqueous phase. The surfactant-rich phase containing concentrated OTG could be directly introduced into the hygro-organic mobile phase of high-performance liquid chromatography with ultra-violet photometric detection. The application of this method greatly enhanced the signal intensity in the chromatogram while reducing the interference of matrix components.

Sensitive densitometry for the determination of platelet-activating factor and other phospholipids in human tears

A sensitive TLC method is reported for the determination of the platelet-activating factor (PAF) and other phospholipids in human tears. Tear samples absorbed on filter-paper were subjected to diatomite column extraction with chloroform-methanol. The eluent containing phospholipids was spotted on a silica gel plate. After removing lipids other than phospholipids by pre-development with hexane-diethyl ether (4 + 1 v/v), individual phospholipid separation was carried out by development with chloroform-methanol-water (65 + 35 + 7 v/v). Phospholipase C and then alkaline phosphatase solutions were sprayed on the TLC plate at 45 degrees C to liberate phosphate from each phospholipid. By spray application of a mixture of ammonium molybdate and Malachite Green, the liberated phosphates appeared as blue-green spots of molybdophosphate-Malachite Green aggregate on a yellow-brown background. The absorbance of each spot on the plate was measured at 620 nm with a densitometer. The peak area was found to be linearly related to PAF content in the range 2-100 pmol per spot, the RSD being 2% (n = 7). Approximately 86% of standard PAF added to tears was recovered. By this method, lysophosphatidylcholine (lysoPC), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human tears could also be detected with high sensitivity. Each phospholipid in healthy human tears showed a nearly constant content; PAF, lysoPC, PC and PE were present at levels of 26.2, 42.3, 10.0 and 19.7%, respectively.

Determination of trace amounts of phosphate in water after preconcentration using a thermally reversible polymer

A method for concentrating and determining phosphate in natural waters, using poly(N-isopropylacrylamide) (PNIPAAm); a thermally reversible polymer, is described. Poly(N-isopropylacrylamide) is soluble in water below 31 degrees C but shrinks abruptly on heating above 31 degrees C, becoming insoluble. With phase separation of PNIPAAm from aqueous solution at 45 degrees C, a molybdophosphate-Malachite Green aggregate (P-Mo-MG), formed by reaction of a Mo-MG reagent, obtained by mixing ammonium molybdate and Malachite Green, was incorporated in PNIPAAm and the resulting solid stuck to the walls of the reaction vessel. After discarding the supernatant solution by decantation, the P-Mo-MG aggregate was dissolved in a small volume of methylcellosolve together with PNIPAAm. The absorbance (lambda max = 627 nm) was proportional to the concentration of phosphate with an apparent molar absorptivity of 2.6 x 10(4) m2 mol-1. This method makes possible the rapid determination of trace amounts of phosphate in water using simple apparatus. The detection limit was 2 nmol dm-3 of phosphate. The method was successfully applied to the determination of phosphate in natural water samples such as tap water, mineral water and rain water.