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Volume 38, Issue 11
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Yukio NAGAOSA, Yukihiro OHTANI1989 Volume 38 Issue 11 Pages 543-546
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSA differential pulse polarographic method was developed for the simultaneous determination of Cu(II) and Fe(III) after extraction of their N-benzoyl-N-phenylhydroxylamine complexes (BPA) into 1:3 ethyl acetate-acetonitrile. Into a polarographic cell, an aqueous sample solution is taken with 2.0 cm3 of 3 mol dm-3 sodium acetate solution, 0.5 cm3 of 20% (w/v) trichloroacetic acid solution and then water to make up a total volume of 4.0 cm3 in a polarographic cell. As the organic solvent, 2.0 cm3 of 1:3 ethyl acetate-acetonitrile solution containing 0.05 mol dm-3 BPA and 0.1 mol dm-3 tetrabutylammonium perchlorate is added to the aqueous solution. The extraction is performed by passing nitrogen through the two phases at a flow rate of ca. 60 cm3 min-1 for 7 min. The differential pulse polarogram is measured in the organic phase under the following conditions: 10 mV s-1 scan rate; 1 s drop time; 50 mV modulation amplitude; 20 ± 0.1°C temperature. The three electrode systems comprise a dropping mercury working electrode, a silver/silver chloride (Ag/AgCl, 3 M KCl) reference electrode and a platinum counter electrode. Two well-defined differential pulse polarographic peaks appear at -0.25 V for Cu(II) and at -0.48 V vs. Ag/AgCl for Fe(III), respectively. The peak heights are directly proportional to the metal concentration in the range of 0.052.0 μg cm -3. The lower limit of detection is 0.02 μg cm-3 Cu(II) and Fe(III) in the original aqueous phase. The method can be applied to the determination of the two metals in biological materials (Bovine Liver, Citrus Leaves and Human Serum).View full abstractDownload PDF (626K) -
Tomoko KITA, Hideyoshi MORITA, Mayumi UMENO, Seizo KITA, Shigeru SHIMO ...1989 Volume 38 Issue 11 Pages 547-552
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSFor the clinical application of ICP-AES, deproteinzation of serum as pretreatment was studied for the determination of nine eliments (Mg, Ca, Fe, Cu, Zn, P, Si, Na and K) in serum by ICP-AES. A mixed solution of nitric acid and trichloroacetic acid was used as the deproteinization solution of serum. To a 0.5 ml portion of serum were added 1.5 ml of water and 4.0 ml of the deproteinization solution. The recommended final concentrations of nitric acid and trichloroacetic acid in the treated serum solution were 0.28 M and 5 %, respectively. After the treated serum solution had stood for 15 min, it was mechanically mixed and then centrifuged at 3000 rpm for 10 min. The elements in the supernatant were then determined by ICP-AES. From a comparison of the results by this method and those by a ICP-AES method conducted in conjunction with a Uniseal Teflon bottle digestion method for pretreatment of serum and those by automatic analytical methods, the present method was shown useful for the determination of elements in serum.View full abstractDownload PDF (943K) -
Hideyoshi MORITA, Tomoko KITA, Seizo KITA, Shigeru SHIMOMURA1989 Volume 38 Issue 11 Pages 553-557
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSTrace element concentrations in the hair of 32 male hemodialyzed patients with chronic renal failure and 33 male healthy persons were measured by multi-channel ICP-AES. Zinc and phosphorus levels were significantly higher than those of the healthy group, while silicone levels were significantly lower than those of the other group. However, erroneous interpretations may occur by a mean-test of element concentrations for the two groups, since many factors can affect hair element content. For example, the relationship between hair color (black and white) and trace element content was demonstrated. From analysis of correlations among the concentrations of the elements, some noteworthy differences between the two groups were found: correlations of Mg-Al (p<0.01), Ca-Al (p<0.001) and Sr-Al (p<0.001)were observed only for the patients, whereas correlations of Ca-Cu (p<0.01), Sr-Cu (p<0.01) and Fe-Al (p<0.01) were found only for healthy persons. However strong correlations (p<0.001) of Mg-Ca, Mg-Sr and Ca-Sr were noted in the both groups. The correlations of Al and other elements for the patient group were differed significantly from those for the healthy group.View full abstractDownload PDF (777K) -
Kenichiro NAKASHIMA, Atuki TAKEYA, Shin'ichi NAKATSUJI, Jyosuke TSUNET ...1989 Volume 38 Issue 11 Pages 558-561
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSThree anthracene derivatives {1-bromomethylanthracene (1-BrMA), 9-bromomethylanthracene (9-BrMA) and 9-chloroacetylanthracene (9-ClAA)} were investigated for their abilities as fluorescent pre-labeling agents for HPLC analysis of saturated aliphatic carboxylic acids. After various conditions such as reaction temperature, reaction time, and reagent concentration were examined, the derivatization procedures were established for ten saturated fatty acids (C4, C6, C8, C10, C12, C14, C1618 and C20). In the next step, separation conditions of the derivatives were studied for a reversed phase column, WAKOSIL 5C-8, with gradient elution using 80100% (v/v) aqueous methanol. According to the conditions established, it was shown that all the fluorescent esters produced by the derivatization of the acids with the three reagents could be separated within ca. 25 min. The detection limits for caproic acid were 100folds lower by using 1-BrMA or 9-BrMA {100 fmol (S/N≥3)} than by 9-ClAA (10 pmol).View full abstractDownload PDF (685K) -
Hitoshi NOHTA, Myungkoo LEE, Tomohiro WATANABE, Hiroaki NAGAOKA, Yosuk ...1989 Volume 38 Issue 11 Pages 562-567
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSA simple and highly sensitive method for the determination of hydroxy radicals by HPLC with fluorescence detection has been developed, based on hydroxylating conversion of phenols (p-coumaric acid, tyramine, octopamine and synephrine) by radicals into catechols (caffeic acid, dopamine, norepinephrine and epinephrine, respectively). After solid-phase extraction using ionexchange cartridges (weak anion type, caffeic acid; strong cation type, the others), catechol compounds formed in hydroxy radicals-generating system (Fe2+/dihydroxyfumarate) and internal standards (3, 4-dihydroxyphenylacetic acid for caffeic acid and isoproterenol for the others) are converted into the corresponding fluorescent compounds by reaction with 1, 2-diphenylethylenediamine. These compounds are separated within 8 min on a reversed-phase column with isocratic elution. The detection limits for the catechol compounds are 1025 fmol in a 100μl injection volume. The amounts of catechols formed and effects of previously reported inhibitors differ according to the phenol used.View full abstractDownload PDF (856K) -
Masaaki KAI, Kazuhiko TAMURA, Hideo WATANABE, Yosuke OHKURA1989 Volume 38 Issue 11 Pages 568-572
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSA fluorometric detection system based on on-line postcolumn reacton with 4-methoxybenzamidine (MBA), a fluorogenic reagent selective for reducing sugars was investigated for analysis of reducing sugars by HPLC. The sugars were separated on a silica gel column (LiChrosorb SI-60, particle size 5 μm) by isocratic elution of acetonitrile, 10 mM tetraethylenepentamine (TEPA) and 50 mM taurine (17:2:1, v/v). The column eluate was mixed with 40 mM MBA and 1.0 M potassium hydroxide and then heated at 100°C for ca. 19 s in a reaction coil (5 m × 0.4 mm i.d., stainless steel tube). The fluorescence from each reducing sugar in the last eluate was monitored at 470 nm (emission) with excitation at 310 nm. The postcolumn fluorescence reaction with MBA was slightly inhibited by TEPA in the mobile phase, but not by acetonitrile or taurine. The proposed reactor system permitted sensitive fluorescence detection for reducing sugars sush as L-rhamnose, L-fucose, D-fructose and D-glucose. Their lower limits of detection (S/N=3) were 2.320 pmol per 100 μl injected. This system was also applicable to other HPLC elution systems using an acetonitrile-water mixture or 0.050.5 M borate buffer (pH 711) as the mobile phase.View full abstractDownload PDF (763K) -
Seiji NAKAMURA, Noriko IMAIZUMI, Kazuichi HAYAKAWA, Motoichi MIYAZAKI1989 Volume 38 Issue 11 Pages 573-577
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSThe detection sensitivities of anionic compounds by conductivity detection (CD) and indirect photometric detection (IPD) were compared in non-suppressed ion chromatography. The reciprocal of peak area to noise ratio was used to compare the two detection methods for such common eluents as benzoate and phthalate at pH 4.3 and pH 6.7. The sensitivities of strong acid compounds were highest by CD at pH 4.3, while those of weak acid compounds were highest by IPD at pH 6.7. These results were consistent with theoretical equations for the two detection methods. The sensitivities of strong acid compounds by IPD exceeded those by CD with the phthalate eluent at pH 4.3, using eluent species such as 1, 5-naphthalenedisulfonate. Correlations between the two methods were excellent in the determination of inorganic anions in soluble fractions of monthly collected deposits.View full abstractDownload PDF (862K) -
Tamotsu WASA, Norihiro NAKATANI, Toshio YAO1989 Volume 38 Issue 11 Pages 578-582
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSAn immobilized salicylate hydroxylase (EC 1.14.13.1; SH) electrode (SHE) and a co-immobilized SH/laccase (EC 1.10.3.2) electrode (SHLE) were prepared by crosslinking with bovine serum albumin and glutaraldehyde on the surface of RVC (reticulated vitreous carbon) impregnated with epoxy resin. The response characteristics of these enzyme electrodes for salicylate were investigated in a phosphate buffer solution (pH 6.0) containing 1 mM nicotinamide adenine dinucleotide (reduced form). The electrode response of SHE and SHLE was measured as an anodic current of catechol produced by SH enzyme reaction and a cathodic current of ο-quinone produced by SH/laccase enzyme reaction, respectively, in a stirred solution (1430 rpm) at 30°C. Linear relationships between current response and salicylate concentration were obtained in a concentration range of 0.1100 μM for SHE and of 0.0110 μM for SHLE. The high sensitivity of the SHLE suggests the occurrence of chemical amplification (about 10 times). The enzyme electrodes were relatively stable and retained about 90% of the maximum activity even following repetitive use for more than 1 month. When not in use, they were stored in a phosphate buffer solution (pH 6.0) at 5°C. SHLE was found applicable to the determination of salicylate in pharmaceutical preparations and the results obtained were in good agreement with indicated values and those obtained by the colorimetric method.View full abstractDownload PDF (814K) -
Methods of steadystate current measurements and voltammetric measurements at higher sensitivityTokuji IKEDA, Fumiyoshi FUSHIMI, Koujiro MIKI, Mitsugi SENDA1989 Volume 38 Issue 11 Pages 583-588
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSQuinoproteins (fructose-, glycerol-, polyamine-, alcohol- and glucosedehydrogenase) were immobilized on carbon paste electrodes by covering the latter with a dialysis membrane. Artificial electron acceptors for the enzymes such as ferricyanide served as mediators for the electron transfer coupling between the electrodes and the quinoproteins immobilized on them. Thus, the quinoprotein-modified electrodes could electrolytically oxidize the substrates. A fructose sensor based on this principle of current response was constructed. Gluconatedehydrogenase, a flavoprotein, from bacterial membranes and ubiquinone were co-immobilized on the basal plane of a pyrolytic graphite electrode by irreversible adsorption. This electrode showed steady-state current response to the substrate due to the electro-enzymic reaction with ubiquinone as a mediator. The electrode was used for voltammetric measurement of the substrate at higher sensitivity, where the reduced form of ubiquinone was preaccumulated by the enzymic reaction prior to electrochemical measurement.View full abstractDownload PDF (906K) -
Yukitaka YAMAMOTO, Toshiharu NUNO, Toshiyuki OSAKAI, Mitsugi SENDA1989 Volume 38 Issue 11 Pages 589-595
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSPolarizable nitrobenzene/water (NB/W) and nitrobenzene containing dibenzo-18-crown-6/water {NB (DB18C6)/W} interfaces function as ion selective electrode (ISE) surfaces for voltammetry (or amperometry), of NMe3H+ and NMe2H2+ ions and of NMeH3+ and NH4+ ions in addition to the above two ions, respectively. The NB/W and NB(DB18C6)/W interfaces were stabilized by covering the interfaces with a hydrophilic dialysis membrane, and the volatile amine sensors were constructed by covering the ISEs based on the membrane-covered NB/W or NB (DB18C6)/W interface with a gas permeable membrane. These volatile amine sensors gave current response proportional to the concentration of NMe3H+ ions using the NB/W interface as ISE and to the sum of the concentrations of NMe3H+ and NH4+ ions using the NB(DB18C6)/W interface as ISE, eventually giving the concentration of NH4+ ions by subtracting the former response from the latter. The pulse amperometric technique was used for these determinations and an aliquot of sample solution was added to the test solution (of pH 10.8), in which the sensor was immersed. The response time was about 100 s. The method was successfully applied to the volatile base nitrogen analysis of fish meats.View full abstractDownload PDF (992K) -
Tsuyoshi NOMURA, Yasuo HIKICHI, Genkichi NAKAGAWA1989 Volume 38 Issue 11 Pages 596-600
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSMagnesium phosphate glasses containing silver oxide show a response for monovalent anions and ammonia with near Nernstian slope. Ammonia and hydrogen peroxide are released by enzymatic reaction of L-amino acids with L-amino acid oxidase. By combining a flow cell composed by this glass membrane and a CPG-10 glass beads column which immobilized with L-amino acid oxidase, L-amino acid can be enzymatically determined with the potentiometric sensor which gives a specific response for liberated ammonia. The chromatographic system employed comprised a HPLC pump, a sintered ceramic filter, a Rheodyne sample injector, a combination of a Toso aminopack column and a treated enzyme column, and a DKK ion-selective flow cell with some improvements. The aminopack column was maintained at 50°C, but the treated enzyme column was operated between 30 and 50°C. When eluents for L-amino acids were applied to 1 mM sodium citrate solutions of pH 5.00, 8.65 and 9.16, these peak heights were linearly related to the concentration of L-amino acids ranging from 10-5 to 5 × 10-3 M, for injections of 100 μl samples. After three months, the treated enzyme column retained about 90% of the original sensitivity. The sensitivity obtained was of the same order as that of the ninhydrin method, while the orthophthalaldehyde method was somewhat more sensitive.View full abstractDownload PDF (709K) -
Shimbu TOMIMURA, Kazuo YASUKOUCHI, Isao TANIGUCHI1989 Volume 38 Issue 11 Pages 601-607
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSEnzyme reactions for sarcosine oxidases from different origins (SOD: from Arthrobacter sp., and SOX: from Bacillus sp.) were analyzed by cyclic voltammetry based on mediated oxidation of the enzymes. The catalytic limiting current observed was used to estimate the enzyme reaction rate(V). The effective mediators for SOD and SOX were not always the same, indicating that the microscopic structures near the redox center of these enzymes would different from each other. Among the mediators examined, octacyanomolybdate acts as an excellent mediator for both SOD and SOX. For the SOD-Mo(CN)84- system, it was observed that the rate (V) shows a hyperbolic dependece on both the sarcosine (S) and the mediator (M) concentrations, giving the Michaelis-Menten type kinetics as V=Vmax/(1+Km1/[S]+Km2/[M]). For the SOX-Mo(CN)84- system, on the other hand, sigmoidal curves were obtained in V vs. [S] and V vs. [M] plots with the rate expression of second-order dependence on both the [S] and [M].View full abstractDownload PDF (929K) -
Kensei KOBAYASHI, Peter E. HARE, Cyril PONNAMPERUMA1989 Volume 38 Issue 11 Pages 608-612
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSSpark discharges in simulated primitive earth atmospheres were carried out, and sugars in the resulting products were analyzed. Pentoses and hexoses were analyzed by GC with a flame thermionic detector (FTD or NPD) and by GC/MS following aldononirile acetate derivatization: The sub-nanogram of each sugar was detected by these methods. When a mixture of methane, nitrogen and water was discharged, no free sugars could be found by GC/MS, though reducing sugars were detected by the Park-Johnson method. When a mixture of methane and water was discharged over a bicarbonate solution with kaolinite, trace amounts of pentoses and hexoses were detected : The total yield of the detected sugars was less than 1 ppm on a carbon basis. The present results suggest that nucleosides may have been produced from pre-formed bases and precursors of sugars, not from free sugars.View full abstractDownload PDF (708K) -
Tadayosi YOSHIMURA, Yukihiro MASUBUCHI, Satoshi OKAZAKI1989 Volume 38 Issue 11 Pages 613-617
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSA new electrochromatoscanning method has been developed for the quantitative analysis of the paper chromatography of catecholamine in food. This method has the characteristic advantages of both paper chromatography and electrochemical quantitative detection. It is simple, convenint, inexpensive and sensitive for organic compound analysis. This paper presents a new approach to the elctrochromatoscanning method for bioanalytical chemistry, and makes possible the quantitative analysis of catecholamine in biological substances, such as bananas.View full abstractDownload PDF (900K) -
Hiroyuki KATAOKA, Mitsuyo MAEDA, Yukizo UENO, Masami MAKITA1989 Volume 38 Issue 11 Pages 618-621
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSA selective and sensitive method has been developed for the determination of O-phosphoethanolamine (PEA) in urine and plasma by GC. PEA was converted to its N-isobutoxycarbonyl dimethyl ester derivative by the procedure reported previously {J. Chromatogr., 436, 67. (1988) }, and measured by GC with flame photometric detection (FPD-GC) using a DB-210 capillary column. By FPD-GC, a linear calibration curve was obtained in the range of 10500 ng of PEA, and the detection limit was about 30 pg as the injection amount. PEA in the urine and plasma samples could be analyzed without any influence from coexisting substances. The recoveries of PEA added to urine and plasma samples were 92105% and the relative standard deviation of the values determined was below 5%. Analytical results of PEA content in urine and plasma samples of normal subjects are presented.View full abstractDownload PDF (597K) -
Shunichi UCHIYAMA, Satoshi KATO, Osamu HAMAMOTO, Shuichi SUZUKI1989 Volume 38 Issue 11 Pages 622-626
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSControlled potential coulometry using electrolyte containing β-D-glucose oxidase, peroxidase and hexacyanoferrate(II) ions was carried out to determine D-glucose selectively. This method has been designated as biocoulometry. D-Glucose added to an electrolyte confined in the pores of porous carbon felt is oxidized by oxygen in the presence of β-D-glucose oxidase, and hydrogen peroxide is produced. Hexacyanoferrate(III) ions produced by the reaction of hydrogen peroxide and hexacyanoferrate(III) ion catalyzed by peroxidase are electrooxidized completely at a -0.5 V vs. counter electrode. The current efficiencies of hydrogen peroxide and D-glucose were found to be nearly 100% and 64%, respectively at pH 5.6 and 22°C. Determination of the time for D-glucose indicated about one minute within 2% of relative standard deviation. This technique was used to determine D-glucose in human, calf and horse sera. The analytical results obtained by coulometry were in fairly good agreement with those by spectrophotmetry. Peroxidase and β-D-glucose oxidases in an electrolytic solution retained their enzyme activity at least for ten days and it was possible to analyze more than 1000 samples using the same electrolyte.View full abstractDownload PDF (699K) -
Chiyo MATSUBARA, Kazuhiro YOSHIHARA, Narumi KOBAYASHI, Yoshiyuki YONEH ...1989 Volume 38 Issue 11 Pages 627-631
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSHigh sensitive spectrophotometric determinations of serum phospholipids were conducted. The levels of serum phospholipids reflect liver functions, so thier determinations are clinically important. Phospholipids are hydrolyzed by phospholipase C and alkakine phosphatase with the release of phosphate. The resulting phosphate associated with Mo-MG reagent (mixed solution of ammonium molybdate and Malachite Green) to form molybdophosphate-Malachite Green aggregates. The aggregates were selectively collected on a nitro cellulose membrane filter (pore size:3.0 μm) and dissolved in methylcellosolve along with the membrane filter. Absorbance (λ: 627 nm) was proportional to the amount of phospholipids. Molar absorptivity of the aggregates in methylcellosolve (ε=2.8 × 105 M-1 cm-1) was large enough to minimize the serum sample for determination. Ascorbic acid, bilirubin, Na+, K+, Ca2+ and Cl- in serum did not affect the results obtained by this method.View full abstractDownload PDF (830K) -
Kenji SHIMADA, Masaharu NAKAJIMA, Hiroyuki WAKABAYASHI, Susumu YAMATO1989 Volume 38 Issue 11 Pages 632-635
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSA simple, sensitive and specific method for the simultaneous determination of mefenamic and flufenamic acids used as anti-inflammatory drugs in human serum by HPLC with electrochemical detection has been developed. To a 20 μl serum sample, 0.9 ml of 0.01 M phosphate buffer solution (pH 5.6) and 8.33 ng of N-phenylanthranilic acid as an internal standard were added. Serum proteins were precipitated with 1 ml of acetonitrile. After centrifugation, the supernatant was concentrated under reduced pressure to dryness using a concentrator with a trap cooled to -70°C. The residue was dissolved in 90 μl of methanol, and 3 μl of that solution were injected onto the column packed with Shimpack CLC-ODS. The elution was performed with a mixed solvent of 1000 ml of 85% methanol containing 0.05 M sodium perchlorate as the supporting electrolyte, and 5.7 ml of glacial acetic acid. The flow rate was 0.6 ml/min. The potential of the glassy carbon as the working electrode was maintained at +1000 mV vs. Ag/AgCl. The recovery of both drugs added to the control serum was satisfactory. The detection limits for mefenamic and flufenamic acids, at a signal-to-noise ratio of 3, were 0.4 pg and 6.3 pg, respectively. The application of the present method to healthy human serum showed mefenamic acid concentrations ranging from 1.2 to 7.5 μg/ml, and flufenamic acid 6.4 to 14.4 μg/ml following the oral administration of mefenamic (125 mg) and flufenamic acid (100 mg) capsules, respectively.View full abstractDownload PDF (602K) -
Masanori TERANISHI, Youichi FUJII, Mitsuru YAMAZAKI1989 Volume 38 Issue 11 Pages 636-642
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESS4-Hydroxyestrogens, which play physiologically important roles, are metabolized to 4-methoxyestrogens by catechol O-methyltransferase. For radioimmunoassay of 4-methoxyestrone (4-MeOE1), antisera were prepared in rabbits by immunization with two types of hapten-bovine serum albumin (BSA) conjugates. The antiserum A was elicited with the C-2 conjugate in which 4-MeOE1 was coupled to BSA by a Mannich reaction. Antiserum B was raised against 4-methoxyestradiol 17-hemisuccinate-BSA conjugate. The antisera A and B bound to approximately 50% of tritium-labeled 4-MeOE1 at final dilutions of 1:47600 and 1:420000, respectively. The association constants of antisera were determined as 6.1×109 and 4.6×1010 M-1 using Scatchard plots. The linearity of standard curve (log-logit plots) ranged from 10 to 1000 pg for antiserum A, while the plots were linear over the range of 2 to 1000 pg for antiserum B. The antiserum A showed cross-reactions to a considerable extent with estrone derivatives substituted at the A-ring. In contrast, the antiserum B underwent no cross-reactions with any compound except 4-methoxyestradiol(107%).View full abstractDownload PDF (907K) -
Atsuo MIYAKAWA, Tohru MAKINO, Akira TAMAGAWA, Kazuho OZAKI1989 Volume 38 Issue 11 Pages 643-649
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSWe constructed an instrument for determining the distribution of intracellular free Ca2+ concentration( [Ca2+]i) using fluorescent Ca indicator Fura-2. This instrument consists of an inverted vertical light fluorescence microscope attached to a 75 W xenon lamp and automatic changer of excitation filters. Image data, recorded by high sensitive SIT camera, are processed by personal computer, and displayed on VDT with pseudocolor. We improved the drug applicator for rapid stimulation of cells and accuracy measurement. The [Ca2+]i estimated with Fura-2 resulted in a lower concentration in comparison with other methods as bioluminescent protein Aequorin, Ca microelectrode, etc. It appears that Fura-2 may interact with intracellular proteins. We used a new Ca concentration buffer containing 80 mg/ml bovine serum albumin (BSA). As for the influence of BSA, fluorescence excitation spectra of Fura-2 alters reduction at 340 nm peak intensity, enlargement at 380 nm, and constant at 360 nm. Also, the appearance dissociation constant of Fura-2 for Ca2+ changed from 109 nM to 138 nM by adding 80 mg/ml BSA. When using the calibration curve calculated by BSA containing the buffer, [Ca2+] i NG108-15 cells with stimulation of 80 mM KCl were increased 185 nM to 1072 nM. These [Ca2+]i almost agreed with the estimated concentration from other methods. Increased [Ca2+]i with KCl stimulation is due to opening voltage sensitive Ca channels present on cell membranes. Fluorescence intensity excited at 340 nm from the KCl stimulated cells increase whole aspect of the cells. But the pseudocolor image showed the peripheral part of the cells to rapidly increase, and then central part to do so slowly. These results due to fluorescence intensity differed with cell thickness that central part of cell show an averaged [Ca2+]i change and peripheral part are a nearly real.View full abstractDownload PDF (3577K) -
Determination of optical purity of (S)-(-)-ofloxacin by HPLC using an opticallly active mobile phaseTakashi ARAI, Hiroko KURODA1989 Volume 38 Issue 11 Pages 650-652
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSHPLC using a chiral mobile phase containing either D-or L-phenylalanine-copper(II) complex was evaluated for purity testing of (S)-(-)-ofloxacin (OFLX). As a result, D-phenylalanine was found to be a suitable chiral additive for optical purity testing of (S)-(-)-OFLX. The detection accuracies obtained with a UV detector at 294 nm and a fluorescence detector at Ex. 330, Em. 505 nm, were compared. It was found that the fluorescence detector could detect with greater stability much less (R)- (+)-OFLX in the presence of (S)-(-)-OFLX than the UV detector. (S)- and (R)-OFLXs in spiked serum were determined simultaneously by connecting a UV detector and a fluorescence detector in series.View full abstractDownload PDF (418K) -
Satoshi KAWAI, Takefumi HIGASHI, Tetsuro FUCHIWAKI, Masafumi TOMITA1989 Volume 38 Issue 11 Pages 652-654
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSMalonaldehyde(MA) reacted with phenylhydrazines to form pyrazole derivatives. The behavior of the three hydrazines, 2, 4-dinitrophenyl-(DNPH), p-nitrophenyl-(NPH) and phenyl-(PhH), in the reaction with MA was compared by HPLC. A Shimadzu LC-5A HPLC equipped with a UV detector (315 nm for DNPH-MA and NPH-MA, 265 nm for PhH-MA) was used for the analysis. A metal column (25 cm×4.6 mm i.d.) was packed with 5-μm Develosil ODS (Nomura Chemical Co., Ltd., Japan). Elution was carried out with CH3CN-0.01 M HCl (40: 60, v/v) at a flow rate of 1.5 ml/min. The reaction of MA with DNPH required a strongly acidic condition, while NPH and PhH reacted well with MA in a weakly acidic medium. The highest peak of the three derivatives was observed from reaction with NPH. The results above showed NPH to be quite useful for estimating a trace amount of free MA in a mild condition.View full abstractDownload PDF (392K) -
Takao YAGI, Yoshindo FUNATO, Naomi ITO1989 Volume 38 Issue 11 Pages 655-658
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSThe simultaneous quantitation of total Mg and total Ca in milk was made by ion chromatography. Total Mg and total Ca were extracted from milk as EDTA complexes. The extract was injected following ultra- or membranefiltration. Non-suppresser type ion chromatography was performed on a Shim-pack IC-Cl cation exchange column (5 mm i. d. × 150 mmL) using 4 mM tartaric acid + 2 mM ethylenediamine (pH 3.41) as the mobile phase. It was found that the coexistance of EDTA·2Na with two equivalents of phosphoric acid disrupted EDTA-Mg (Ca) complexation. If sample pH is ≤10, and total Mg (Ca) in the EDTA complex does not exceed 1 μg, the dissociation of the EDTA complex to detectable Me2+ (Ca2+) cations is mobile phase pH-dependent. Typical milk samples (n=4) yielded 112.5 115.7 ppm total Mg, and 1151.5 1190.6 ppm total Ca. Comparison with values obtained from AAS (=100%) showed 106% and 103% for Mg and Ca, respectively.View full abstractDownload PDF (510K) -
Hiroyuki UKEDA, Miho MATSUBARA, Kiyoshi MATSUMOTO, Yutaka OSAJIMA1989 Volume 38 Issue 11 Pages T165-T169
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSNADH formed in the reaction of alcohol dehydrogenase (ADH) was regenerated to NAD with dissolved oxygen using vitamin K3 and diaphorase (DI). The regeneration method makes it possible to construct an FIA system for ethanol by using a Clark oxygen electrode detector. The phosphate buffer (0.05 M, pH 8.0) carrier solution was pumped through a sample injection valve and a reactor packed with Sepharose on which ADH and DI were co-immobilized, and transported to a new type of flow-through cell in which the surface of the oxygen electrode was set upward into the carrier stream. The peak current was linearly related to ethanol in the range 4 × 10-4 M to 2 × 10 -3 M. The detection limit was 5 × 10-5M and the R.S.D. was 1.1% for 5 successive determinations at 2 × 10-3 M level. The sampling frequency was 15 samples/h. When the system was applied to the determination of ethanol in wine, the results were in good agreement with those obtained by the F-kit method.View full abstractDownload PDF (714K) -
Keiji GAMOH, Hiroyuki MURAKITA, Tomio FUJITA1989 Volume 38 Issue 11 Pages T170-T173
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSWe present a rapid and conventional HPLC method for the simultaneous determination of anticonvulsants, ESM, PM, PB, CBZ, PHT, NZP, CZP and DZP in serum using the dual column-dual detection technique. The system consists of two flow lines connected through the flow changeover valve, and is controlled by a time program using a microprocessor. These compounds were separated by the reversed phase mode with isocratic elution. The analytical procedure was as follows; a serum extract was injected into the first analytical column. After 0.5 min, the flow line was changed to the dotted line by turning the changeover valve. In this stage, weakly retained compounds, such as ESM, PM and PB were eluted into the second column, and monitored at 220 nm. After 7 min, the flow line was returned to the original line. CBZ, PHT and benzodiazepines were separated in the column 1 and monitored at 254 nm. Both detectors 1 and 2 are connected to data processor and thus analytical results can be calculated at the same time. A single analysis was completed in about 20 min. The calibration curves for ESM and NZP were linear over the ranges 5.768.4 μg/ml and 17160 ng/ml, respectivery. Recovery of the drugs from spiked serum were from 98.9±0.9% (ESM) to 78.6±4.9 (NZP). The present method was used for the determination of anticonvulsants in the serum of an epileptic patient.View full abstractDownload PDF (548K) -
Kenji MATSUYAMA, Choichiro MIYAZAKI, Tatsuya YAMASHITA, Masataka ICHIK ...1989 Volume 38 Issue 11 Pages T174-T178
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSA fluorometric HPLC method for the determination of valproic acid (VPA) in mouse brain was developed using 3- bromomethyl-6, 7-dimethoxy- 1-methyl-2 (1H) -quinoxalinone(Br-DMEQ) as a prelabelling reagent. The method was used to study the interrelations among the concentrations of VPA, γ-aminobutyrate as an inhibitory amino acid and glutamate or aspartate as an excitatory amino acid in mouse brain. The brain levels of VPA decreased with a half life of 0.99 h and attained almost zero at 5 h after the intraperitoneal administration of VPA at a dose of 200 mg/kg to mice. The administration of VPA gave rise to a significant increase in γ-aminobutyrate in response to a significant decrease in aspartate and glutamate in mouse brain. In particular, aspartate continued to decrease up to 8 h after administration whereas VPA disappeared at 5 h. The present observations are considered closely correlated to the “carried over effect” of VPA.View full abstractDownload PDF (619K) -
Yasuhiro NAKAHARA, Yuko YOSHIOKA, Masahiro MURATA, Syuhei KURASHINA, K ...1989 Volume 38 Issue 11 Pages T179-T182
Published: November 05, 1989
Released on J-STAGE: June 30, 2009
JOURNAL FREE ACCESSWe have compared the serum theophylline levels determined by Vision® Analyzer and fluorescence polarization immunoassay (FPIA). Eighty-five blood samples were collected and the serum was analyzed for theophylline using both methods. The equation for the regression line is Visio®=0.88 × FPIA+1.21, r=0.97. Total bilirubin at 17.2 mg/dl and hemoglobin at 230 mg/dl caused interference that was detected by the Vision® Analyzer. Serum theophylline determined on Vision® is precise and agrees with an established procedure.View full abstractDownload PDF (460K) -
Yasushi HAKETA, Ryoichi MOTOHASHI, Kazuhito KAJIMARA, Naoki MATSUNAGA, ...1989 Volume 38 Issue 11 Pages T183-T186
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSAn on-line FIA system was developed for intracellular ATP measurement in a culture fluid and measurement conditions were examined. This system was then used for intracellular ATP measurement in microorganisms. ATP concentration was measured by bioluminescent reaction using luciferase and luciferin. Only about 5 min were required to measure intracellular ATP and free ATP concentrations. Luminescence response was linear from 1 nmol/l to 10 μmol/l. Measurement time could be over 7 d long to preserve the enzyme solution and ATP standard at 4 °C in the cooling container. Intracellular ATP was measured in culture fluids of yeast and E. coli. There was good correlation between intracellular ATP concentration and viable cells number. For yeast cells, the correlation coefficient and R.S.D. were 0.992, lower than 3%. The results of the present system agreed with conventional one well.View full abstractDownload PDF (640K) -
Naoshi FUJIWARA, Tatsuhiko YUASA, Koki SHIMOJI1989 Volume 38 Issue 11 Pages T187-T190
Published: November 05, 1989
Released on J-STAGE: February 16, 2010
JOURNAL FREE ACCESSGlucose metabolism in the rat brain was investigated by 13C-NMR spectroscopy. Rats were intravenously infused with [1-13C]glucose. 13C-NMR spectra were measured by a gated 1H-decoupling method without nuclear overhauser effect. Infusion of [1-13C]glucose gave apparent signals of glucose-Cl carbon in the α- and β-anomer forms and those of C2, C3 and C4 carbon in glutamate. During hypoxia, the signal of lactate-C3 increased, and the α-glucose signal diminished while the β-glucose signal maintained its intensity. These changes in 13C signals reflect glucose metabolism in the brain. Although the amplitudes of signals do not directly correspond to the concentrations of metabolites, chemical and other information on biological substances could be obtained in living systems by in vivo 13C-NMR spectroscopy.View full abstractDownload PDF (568K)
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