Kazushige Yokota

A heterologous enzyme immunoassay of prostaglandin E2 using a stable enzyme-labeled hapten mimic

A sensitive heterologous enzyme immunoassay for prostaglandin E2 was developed using 9-deoxy-9-methylene-prostaglandin F2 alpha as a stable prostaglandin E2 mimic. beta-Galactosidase was conjugated to the hapten mimic. Anti-prostaglandin E2 IgG was bound to a polystyrene tube. The enzyme-labeled hapten mimic mixed with unlabeled prostaglandin E2 was allowed to react in a competitive manner with the immobilized antibody. Then, the beta-galactosidase specifically bound to the antibody was assayed fluorometrically, and the enzyme activity was correlated with the amount of unlabeled prostaglandin E2. According to the calibration curve thus obtained, prostaglandin E2 could be determined in a range of 1.2-430 fmol. Prostaglandin E2 was extracted from human urine by the use of an octadecylsilyl silica column. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high content of prostaglandin E2. The interfering substance was separated from prostaglandin E2 by reverse-phase high-performance liquid chromatography. The purified urinary extract was examined by the enzyme immunoassay for prostaglandin E2, and the validity of the results was confirmed by gas chromatography-selected ion monitoring.

Possible induction of fatty acid cyclooxygenase in mouse osteoblastic cells (MC3T3-E1) by cAMP

Prostaglandin E2 (PGE2), a bone-resorption factor, was essentially the sole arachidonate metabolite in an osteoblastic cell line cloned from mouse calvaria (MC3T3-E1). When the cells were cultured in the presence of 2% newborn bovine serum, 1 microM epinephrine markedly stimulated PGE2 synthesis from endogenous arachidonic acid. The PGE2 synthesis commenced after a lag phase of 1-2 h, and reached a maximum at about 3 h after the addition of epinephrine. The effect of epinephrine was inhibited by propranolol, and epinephrine could be replaced by isoproterenol, suggesting beta-adrenergic stimulation of PGE2 production. A rapid increase in intracellular cAMP was observed upon the addition of epinephrine. When the intracellular cAMP level was raised using cholera toxin or forskolin, the PGE2 synthesis was also stimulated. The enhanced PGE2 synthesis was attributed to an increased level of cyclooxygenase, which was shown by immunoprecipitation of the enzyme using anti-cyclooxygenase antibody. Inhibitors of transcription and translation suppressed the epinephrine-dependent increase in cyclooxygenase activity. These findings suggest induction of cyclooxygenase involving cAMP via an as yet unclarified mechanism.

Enzyme immunoassay of thromboxane B2

An immunoassay for thromboxane B2 was developed in which the hapten molecule was labeled with beta-galactosidase. The immunoprecipitate formed after competition between enzyme-labeled and unlabeled thromboxane B2 was subjected to a fluorometric assay of beta-galactosidase. Thromboxane B2 was detectable in the range of 0.1-30 pmol. Both enzyme immunoassay and radioimmunoassay showed essentially the same cross-reactivities with other prostaglandins and their metabolites when the same antibody was used. Known amounts of thromboxane B2 were added to human plasma, and the sample was applied to an octadecyl silica column. The extract was analyzed by enzyme immunoassay to examine the correlation between the added (x) and measured (y) thromboxane B2 (y = 1.09x + 11.07 pmol/ml, r = 0.99). A satisfactory correlation was observed between radioimmunoassay (x) and enzyme immunoassay (y) (y = 0.92x + 4.64 pmol/ml, r = 0.96). The validity of enzyme immunoassay was also confirmed by gas chromatography-mass spectrometry of a dimethylisopropylsilyl ether derivative of thromboxane B2 methyl ester. The method was applicable to the assay of thromboxane B2 produced from endogenous precursor during thrombin-induced aggregation of human platelets.

Enzyme immunoassay of 6-ketoprostaglandin F1α in a solid phase

Abstract A solid-phase enzyme immunoassay of 6-ketoprostaglandin F1α (a stable degradation product of prostaglandin I2) was developed in which the hapten molecule was labeled with β-galactosidase. The antiserum was bound to a polystyrene tube, and the enzyme-labeled and unlabeled 6-ketoprostaglandin F1/ga were allowed to react in a competitive manner with the immobilized antibody. The activity of the immunologically bound β-galactosidase was assayed by fluorometry, and correlated with the amount of unlabeled 6-ketoprostaglandin F1α According to a calibration curve 6-ketoprostaglandin F1α was detectable in a range of 30 fmol-10 pmol. When 6-ketoprostaglandin F1α was extracted from human serum by using an octadecylsilyl silica column (Sep-Pak C18) and the crude extract was subjected to the enzyme immunoassay, the content of 6-ketoprostagland F1α was 50–60 pmol / ml of human serum. An endogenous substance(s) which disturbed the immunoreaction and gave such an apparently high concentration of 6-ketoprostaglandin f1α was separated from the endogenous 6-ketoprostaglandin F1α by HPLC. With the purified 6-ketoprostaglandin F1α fraction there was a good correlation (r = 0.994) between enzyme immunoassay and radioimmunoassay. The validity of the enzyme immunoassay was confirmed by gas chromatography-selected ion monitoring.

Gas chromatography—mass spectrometry of some prostanoids with new derivatizing agents : Application to the analysis of 13,14-dihydro-15-ketoprostaglandin F2α

Abstract 13,14-Dihydro-15-ketoprostaglandin (PG) F 2α was separated completely from PGD 2 , PGE 2 and PGF 2α , within 10–15 min by gas chromatography—selected-ion monitoring on a capillary column after conversion to their methyl ester— n -butyloxime—dimethylisopropylsilyl (ME—nBO—DMiPS) ether or ME—nBO—cyclic diethylsilylene (DES) derivatives. The mass spectra were characterized by their inherent ions of [M — 43] + at m/z 596 for the ME—nBO—DMiPS ether and of [M — 73] + at m/z 450 for the ME—nBO—DES derivatives, respectively. The appearance of the characteristic ions in the high-mass region with relatively high intensity and the use of these ions in selected-ion monitoring in a high-resolution mode ( M/ΔM = 3500) made it possible to enhance the selectivity in the detection of 13,14-dihydro-15-keto-PGF 2α in biological fluids. The detection limit of the ME—nBO—DES derivative of 13,14-dihydro-15-keto-PGF 2α was found to be 2 pg with a signal-to-noise ratio of 4:1 when the ion of [M — 73] + was monitored at m/z 450.30.

Lipoxygenase-Quercetin Interaction: A Kinetic Study Through Biochemical and Spectroscopy Approaches

Lipoxygenases (EC 1.13.11.12, linoleate:oxygen, oxidoreductases, LOXs) which are widely found in plants, fungi, and animals, are a large monomeric protein family with non-heme, non-sulphur, iron cofactor containing dioxygenases that catalyze the oxidation of polyunsaturated fatty acids (PUFA) as substrate with at least one 1Z, 4Z-pentadiene moiety such as linoleic, linolenic and arachidonic acid to yield hydroperoxides (Gardner, 1991).

Interactive Pro-Adipogenic Effect of 15-deoxy-Δ12,14-Prostaglandin J2 to Interfere the Inducible Synthesis of Anti-Adipogenic Prostanoids in Cultured 3T3-L1 Preadipocytes, an Important Molecular Event to Consider for Drug Development

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh; Center for Integrated Research in Science, Shimane University; United Graduate School of Agricultural Sciences, Tottori University; Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University; Department of Clinical Pharmacy, Faculty of Pharmacy, Tokushima Bunri University