Capillary electrophoresis (CE) combined with laser-induced fluorescence detection is applied to the determination of amino acids in urine samples. The urine samples are first ultrafiltered, to remove proteins and large peptides, and the filtrates are then directly labeled by reaction with fluorescein isothiocyanate (FITC). Cyclodextrin-modified CE using alpha-cyclodextrin is employed for the separation of the FITC-labeled amino acids. Seven amino acids are clearly separated from side reaction products produced during the labeling reaction, when an 80 mM borate buffer containing 45 mM alpha-cyclodextrin is used as the running buffer. For quantitative analysis, rhodamine B is added to the labeled urine samples as an internal standard. The calibration curves for phenylalanine, glutamine, proline, glycine, serine, alanine, and valine are linear in the range of 10 microM to 100 microM. The concentration limits of detection for all of the amino acids are estimated to be 160-330 nM. Conversely, the limit of quantitation (LOQ) was approximately 10 microM and the limitations are due to the labeling efficiency rather than the sensitivity of the detector. Three amino acids in urine samples, glutamine, glycine, and alanine, are readily quantitated, while the concentrations of the others are below the LOQ. The present method would permit the determination of seven amino acids in urine successfully.

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Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the interaction between androgen receptor (AR) tagging of a green fluorescent protein (GFP) and the ligands in living cells. The fluorescence lifetime of the AR-GFP without ligands was ca. 3.1 ns, which was reduced to ca. 2.5 ns after treatment with agonist 5α-dihydrotestosterone. On the other hand, the fluorescence lifetime of AR-GFP was not changed after treatment with antagonist hydroxyflutamide. The reaction kinetics was simulated in the present study, and the obtained results indicated the possibility of the presence of an intermediate complex during the reaction. FLIM can be used to record the ratio of the AR as it reacts with an agonist, and, therefore, it is useful for acquiring information concerning the interaction between AR and ligands in living cells.Key words androgen receptor; 5α-dihydrotestosterone; hydroxyflutamide; fluorescence lifetime imaging microscopy; green fluorescent protein Fluorescence intensity imaging microscopy has been widely used in cytology and histology. However, fluorescence intensity images cannot quantitatively be compared with each other, since fluorescence intensity is deduced as a relative value. On the other hand, fluorescence lifetime is measured as an absolute, and is influenced by the characteristics of a fluorophore's microenvironment, such as temperature, viscosity, and pH. Therefore, it is possible to use fluorescence lifetime imaging microscopy (FLIM) to evaluate biological phenomena in living cells.1,2) We have recently developed a fluorescence lifetime imaging microscope that consists of a picosecond dye laser and a time-gated intensified charge-coupled device (ICCD) camera. 3,4) In this study, the FLIM system was applied to the monitoring of an androgen receptor (AR) with tagging green fluorescent protein (GFP) in living cells. AR is one of the steroid hormone receptors present in the cytoplasm without ligands, and it is subsequently translocated into the nucleus after binding.5) Until now, the location of AR in living cells has been observed primarily through the use of fluorescence intensity images. [6][7][8] We confirmed the intracellular location of AR-GFP after treatment with 5α-dihydrotestosterone (DHT) and a hydroxyflutamide (OH-FLU) by using the FLIM system. Moreover, the results of the dose-dependent experiment were also simulated in the present study. ExperimentalCell Culture and Transfection of AR-GFP COS-7 cells were obtained from the Japanese Collection of Research Bioresources, and were maintained in Dulbecco's minimal essential medium (DMEM; Invitrogen, U.S.A.) supplemented with 10% fetal bovine serum (FBS; Gibco, U.S.A.) and AntibioticAntimycotic at 37°C and 5% CO 2 .The AR-GFP plasmids (pEGFP-N2) were kindly provided by H. Iwamoto (Kyushu University). The cells were cultured in 35 mm glass-bottom dishes (Mat-Tek) (5×10 4 cells/dish) on the day before transfection. The AR-GFP plasmids and transfection reagent (Superfect transfection reagent, Invitrogen) were added to 100 µL DMEM ...