Fumio Iinuma

Determination of quinolinic acid in human urine by liquid chromatography with fluorimetric detection

Abstract A system has been developed for the determination of quinolinic acid in human urine using liquid chromatography with fluorimetric detection. The compound is separated by reversed-phase chromatography using a 35 mM potassium dihydrogen-phosphate solution (adjusted to pH 3.8 with 0.2 M citric acid) containing 350 mM hydrogen peroxide and 0.05 mM tetramethylammonium hydroxide as a mobile phase. The compound in the column effluent is irradiated with ultraviolet light to produce fluorescence. This fluorescence is monitored with excitation at 326 nm and emission at 380 nm. The calibration graph for quinolinic acid is linear over the range 0.36–68.8 nmol ml−1 upon injecting 10 μl of the standard solution. Pretreatment of urine was achieved by dilution and filtration only. The mean recovery of quinolinic acid from urine is more than 95%.

Sensitive determination of melatonin by precolumn derivatization and reversed-phase high-performance liquid chromatography.

A sensitive determination method for melatonin was developed. Melatonin was derivatized under alkaline conditions in the presence of hydrogen peroxide. The resultant fluorophore was excited at 247 nm and the emission wavelength was 384 nm. The Stokes shift was 137 nm, which was longer than that of melatonin itself (lambda ex 280 nm, lambda em 330 nm). The melatonin derivative was separated by reversed-phase HPLC in about 15 min and the calibration curve was linear from 500 amol to 5 pmol (r > 0.999) with the detection limit of 500 amol (S/N = 5). The sensitivity of this method was about ten times higher than that of previous methods. Both the day-to-day precision and within-day precision were about 5%, and the derivative of melatonin in the aqueous solution was stable for more than 10 days. This method was successfully applied to the determination of melatonin in rat pineal gland.

Fluorimetric determination of isatin in human urine and serum by liquid chromatography postcolumn photoirradiation

For the fluorimetric determination of isatin in human urine and serum, HPLC-postcolumn photoirradiation using a mobile phase has been developed. Isatin in the urine or serum sample was separated on a Capcell Pak C1 column (250 x 4.6 mm id). The mobile phase consisted of 70 mmol l-1 phosphate buffer (pH 7.2)-tetrahydrofuran (85 + 15% v/v) containing 5 mmol l-1 hydrogen peroxide, which was irradiated with germicidal light to induce fluorescence (lambda ex 302 nm, lambda em 418 nm). The addition of tetrahydrofuran to the mobile phase led to the peaks showing good separation as well as increased sensitivity. The calibration graph for isatin was linear over the range of 0.16-10.7 ng. The pretreatment of the acidified urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively. The mean recovery of isatin from urine and serum was greater than 94%.

Determination of disodium cromoglycate in human urine by high-performance liquid chromatography with post-column photoirradiation-fluorescence detection

For the determination of disodium cromoglycate in urine, a fluorimetric method using HPLC post-column photoirradiation has been developed. The mobile phase consisted of a 35 mmol l-1 phosphate buffer (pH 8)-methanol (7 + 3, %v/v) containing 75 mmol l-1 hydrogen peroxide and 20 mmol l-1 18-crown-6. The 18-crown-6 was used for separation adjustment of the disodium cromoglycate in the urine sample. Photoirradiation was carried out in tubing wound around a germicidal light in a reactor equipped with an air-cooling fan. The fluorescence was monitored with excitation at 325 nm and emission at 448 nm. The calibration graph for disodium cromoglycate was linear over the range 38-2340 ng ml-1 using an injection volume of 100 microliters. The pretreatment of the urine samples consisted of diluting and filtering steps. The mean recovery of disodium cromoglycate from urine was 99.1 +/- 2.4% (n = 6).

Fluorimetric determination of nicorandil in human plasma by a high-performance liquid chromatographic-postcolumn ultraviolet detection system equipped with on-line back-pressure tubing

Abstract For the determination of nicorandil in plasma, a fluorometric technique using HPLC-postcolumn UV detection has been developed. The chromatographic system consisted of a single pump, photoreactor and on-line back-pressure tubing. The system was suitable for the separation of nicorandil under the present reaction conditions. The calibration graph was linear over the range 6.5–1170 ng ml−1 using an injected volume of 100 μ1. The pretreatment of the plasma samples consisted only of deproteinizing steps by adding perchloric acid. The mean recovery from plasma was 90.2%.

Induction of CD4+ regulatory T cells by 12-O-tetradecanoylphorbol 13-acetate in mice. Part 1: The strain difference in this induction.

12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of signal transduction, induced antigen-nonspecific regulatory T (Tr) cells for delayed-type hypersensitivity (DTH) in the spleen. A marked strain difference in the induction of Tr cells was observed when the study was performed by using the several strains of mice at 6-8 weeks old. TPA painting induced CD4+Tr cells in C3H/He (H-2k), C3H/HeN (H-2k), DBA/2 (H-2d) and AKR/N (H-2k) mice, but not in C57BL/6 (H-2b), C57BL/10 (H-2b), BALB/c (H-2d) and A/J (H-2a). Regulatory cells were also induced by incubating spleen cells from unprimed mice with TPA in vitro and were seemed to act by the production of soluble factor(s). A downregulatory activity of the soluble factor(s) was abrogated by SXC-1 (anti-IL-10 monoclonal antibody), but not by SXC-2 (anti-IL-10 monoclonal antibody) and anti-IL-4. For purification of the factor(s), we established the T cell hybridoma 4C5-1 by the fusion of spleen cells from TPA-treated C3H/He mice with AKR thymoma Bw5147 cells. The 4C5-1 cells secrete the factor(s) which can inhibit DTH response. The inhibitory activity of the factor(s) could be neutralized by SXC-1, but not by SXC-2, anti-IL-4, anti-IL-6 and anti-TGF-beta antibodies. The factor(s) could not affect the proliferation and IFN-gamma production of alpha s1-casein-specific 3D20 Th1 cells. The factor(s) termed DIF (DTH Inhibitory Factor) may be a novel cytokine, since they have reduced the footpad swelling response by local injection, and have no immune crossreactivity with the DTH regulatory cytokines and no inhibitory activity for in vitro Th1 response.

Induction of CD8+ suppressor T cells by treatment with DMBA and TPA in vitro

Antigen-nonspecific CD8+ T suppressor cells, which suppressed delayed-type hypersensitivity (DTH) against sheep red blood cells in BALB/c mice, were induced by incubating spleen cells from mice treated with 7,12-dimethylbenz[a]anthracene (DMBA), a tumor initiator, with 12-O-tetradecanoylphorbol 13-acetate (TPA), a tumor promoter. The optimal condition was incubation in 3.2 x 10(-8) mol/5 ml of TPA for 4 days. It was shown that induction of the suppressor cells required macrophages from mice treated with DMBA. These data were consistent with the results of previous work, in which CD8+ suppressor cells were induced by painting BALB/c mice with TPA following DMBA treatment. DTH was suppressed in the culture supernatants of spleen cells from mice treated with DMBA and TPA; the suppression was genetically unrestricted. The suppressor factor was resistant to trypsin and sensitive to heating at 56 degrees C for 30 min and had affinity for the macrophages.

Xanthurenic acid in the mutant strain scarlet of Drosophila melanogaster

Abstract 1. 1. Xanthurenic acid, 3-hydroxykynurenine, and kynurenine hydroxylase activity in bw, st and bwst of D. melanogaster were determined. 2. 2. Both pupa and adult of bw, st and bwst showed much the same level of kynurenine hydroxylase activity. 3. 3. The pupae of bw, st and bwst contained not a little 3-hydroxykynurenine. In st and bwst , the 3-hydroxykynurenine rapidly decreased at emergence, while in bw , the decrease was rather gradual. 4. 4. In bw and st , a considerable amount of xanthurenic acid was synthesized during the pupal stage, while, in bwst , the synthesis was moderate. Moreover, in the adult stage, xanthurenic acid was much less in bwst than in bw and st .