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.
The introduction of a 16 alpha-hydroxyl function into the steroid nucleus was studied in resting cells of Streptomyces roseochromogenes NRRL B-1233. The oxidation product of dehydroepiandrosterone (DHEA) was identified as 16 alpha-hydroxy DHEA by using thin-layer and gas-liquid chromatography. A linear relation between cell concentration and 16 alpha-OH-DHEA formation was observed. 16 alpha-Hydroxylase showed good activity at pH 8.0 for 16 alpha-OH-DHEA formation. The enzyme showed good activity at 3.1 x 10(-4) M DHEA. The oxidation products of pregnenolone, 4-androstene-3,17-dione, estrone, and 5-androstene-3 beta,17 beta-diol as well as of other substrates were identified as the 16 alpha-hydroxy steroid, respectively. The rates of microbial 16 alpha-hydroxylation were as follows: 76.9% for DHEA, 50.4% for pregnenolone, 43.9% for 4-androstene-3,17-dione, 34.3% for estrone, and 19.6% for 5-androstene-3 beta,17 beta-diol. The organism tested catalyzes 16 alpha-hydroxylation of a wide variety of steroids.
Microbial introduction of a 16whydroxyl function into the steroid nucleus (EiuLqeqcrtcqrii u t i i 18. 12. JYih') Tlie iritiodrrction of a 1Ka-liydroxyl function into t h r strroid nucleiis n as studied in resting ~~~1 1 s o f Ntrrpfonryces roseochrotnogenes N RKI, l3-1233. The oxidation product of dehydroepiandrostt~r~ri~, (DHEA) u as identified as 16a hydroxy DHEA by using thin-!ayer and gas-liquid chromatograph). A linear relation between cell concentration and 1Ga-OH-DHEA formation uas observed. 16a-Hydroxylase showed good activity at pH 8.0 for lea-OH-DHEA formation. The enzyme shomcti , I 1 Iic ositl~itioii products xc.rc. obtainctl fro111 tlic c~riltrirvs after I5 Ilr of iticiilia tiuu. :) GC'L analysis -\\-as performed wit11 a HLT.S(.HI 163. using n 2 ni ; i 3 niin J ) Solrent systcni: Chloroform: methanol (97: 3, v / v ) column packed \vith 1.50& OV-1
A 4-years-old boy is reported. At age 1 he received Bacille Calmette-Guêrin (BCG) vaccination and a month later developed swelling and flaring of the inoculated site and ipsilateral axillary lymph node enlargement (diameter 30 mm). He was diagnosed BCG induced lymphadenitis and oral INH was administered for 6 months. After 2 years, chest roentgenogram revealed calcification of the involved axillary lymph node. After 2 and half years lymph nodes showed decrease in size and he was considered to be cured. However, after 3 years on May/30/ 1996, he presented enlargement of lymph node on the same site. He took Rubella vaccine 50 days prior and DPT booster vaccine 8 days prior. Chest CT revealed enlarged multiple lymph nodes with calcification. Acid fast bacteria was detected in the lymph node biopsy specimen under light microscope and diagnosis of tuberculous lymphadenitis was obtained pathologically. However lymph node culture did not prove tubercle bacillus and PCR analysis for DNA of Mycobacterium tuberculosis was negative. The patient was immunocompetent by history and examination for immunological function was normal. It was suspected from clinical course that BCG lived in the lymph node and recrudesced after 3 years following vaccination.
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