PhaR fromcoli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).

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In this study, we established a method for the quantitative measurement of native adrenal steroids with GC-MS equipped with capillary column (cross-linked methyl silicone 25 m X 0.2 mm I.D., 0.11 m thin film). 1 ml of serum sample containing 5 alpha-cholestane as internal standard (IS) was elicited by organic solvent using extrelunt column. These samples were derived by n-butylboronic acid, o-methylhydroxylamine and trimethyl-silylating agents, then were finally applied to GC-MS. The intensities of molecular ions were used for the measurement of the serum concentration of steroids. The molecular ion peaks of steroids were obtained at m/z460 (17 alpha-hydroxyprogesterone; 17OHP), m/z548 (corticosterone; B), m/z470 (11-deoxycortisol; S), m/z417 (pregnenolone; PL), m/z372 (progesterone; PT), m/z558 (cortisol; F), m/z389 (dehydroepiandrosterone; DHEA), m/z371 (estrone; E1), m/z416 (estradiol; E2), m/z504 (estriol; E3), m/z389 (testosterone; T), m/z344 (androstenedione; A) and m/z372 (IS). The curve of calibration for each steroid showed good linearity. The sensitivities of the GC/MS method were less than 5pg/one shot of each sample. The coefficients of variations of accuracies and precisions in this GC/MS method were less than 15% of each steroid. The samples from normal subjects after metyrapone and ACTH loading tests, and the patients of congenital adrenal hyperplasia showed a good correlationship between the data of GC/MS and the data of RIA after sephadex LH-20 column-chromatography. These results implied the usefulness of our system in clinical application. Moreover, this assay takes only 3 hrs. Thus it saves much time in comparison with the time-consuming radioimmunoassay system.

together with 2 ml of 0.1N HC1 for 15 minutes.After we discarded the ethyl acetate layer, the residual HC1-layers were alkalinized again at a pH above 7.8 with 0.3N NaOH and were again extracted three times with ethyl acetate.The ethyl acetate layers containing metyrapone, reduced metyrapone and the internal standard were evaporated to dryness with a stream of nitrogen in a 37°C water bath.Following the above procedures, reduced metyrapone was converted to a trimethylsilyl (TMS) derivative of reduced metyrapone in the mixture (TMCS : TMSI : BSTFA = 3:1:3) for the determination of serum reduced metyrapone. This silylated reduced metyrapone at C-1 position improved tailing on the gas chromatogram.The final samples, containing metyrapone, TMS derivatives of reduced metyrapone and the internal standard, were subsequently applied to GC/MS.The conditions of the GC/MS procedure were as follows: The glass column was packed with 3% OV-17 chromosorb W (HP) 80-100 mesh, the injection port was set at 270°C, the column temperature was 220°C, the ionization voltage was 70eV, the accelerating voltage was 3KV. Electron impact ionization was used for the measurement.The areas of mass fragmentgram, measured at m/z 106 for metyrapone, m/z 180 for TMS-derivative of reduced metyrapone and m/z 136 for the internal standard, were used for the calculation of the serum concentrations of metyrapone and reduced metyrapone.The calibration curve showed good linearity (7=0.999; P<0.001) from 20 pg to 500 pg. The final recoveries of metyrapone and reduced metyrapone from serum were found to yield 90 ± 5% for metyrapone and 85 ± 6% for reduced metyrapone.The coefficients of variation of accuracy and precision were less than 8 .40% in the range of 20 pg to 500 pg.The lower limits of sensitivity to metyrapone and reduced metyrapone in this assay system were 20 pg/one shot (S/N > 20), respectively.The serum metyrapone and reduced metyrapone concentrations were analyzed pharmacokinetically, according to the one-compartment open system model, especially with regard to age-specific differences.The pharmacokinetic values after oral and rectal administration of metyrapone were statistically proved to show no significant differences either among the various age-groups or due to the routes of metyrapone administration in children. However, significant differences were observed with regard to the ratio of the fraction of the administered dose of metyrapone to the apparent distribution volume of metyrapone (F/V). Since the parmacokinetic values of serum metyrapone metabolism of various age-groups were not found statistically different, these pharmacokinetic values could all be included in the same control group.The pharmacokinetic mean values of metyrapone metabolism in all groups were as follows: the apparent first-order absorption rate constant was 1.329 h1, the apparent firstorder rate constant for renal excretion + bioactive and non-bioactive metabolite formation 第59巻 第5号