Detection of Mycobacterium tuberculosis in clinical specimens by polymerase chain reaction method.

An insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis was used as a target for amplification using the polymerase chain reaction (PCR) assay for detecting Mycobacterium tuberculosis in clinical specimens. The sequences of primers were 5'-CCTGCGAGCGTAGGCGTCGG-3' (primer 1) and 5'-CTCGTCCAGCGCCGCTTCGG-3' (primer 2). One cycle of amplification consisted of denaturing at 94 degrees C for 2 min, primer annealing at 68 degrees C for 2 min, and extension at 72 degrees C for 2 min. DNA (5 fg) extracted from M. tuberculosis was detected by gel electrophoresis and Southern blot hybridization after 40 cycles of amplification. The amplification products were not obtained by DNA extracted from M. kansasii, M. intracellulare, M. avium, M. fortuitum, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Legionella pneumophila and Staphylococcus aureus; only from the M. tuberculosis complex. PCR results were compared with conventional cultural, pathological and microscopic findings in the detection of M. tuberculosis in 112 clinical specimens. There were 25 specimens that were positive for M. tuberculosis by cultural or pathological examination, of which 20 (80%) were positive by PCR. PCR detected the organism in 5 (83%) of 6 smear-positive specimens and 15 (79%) of 19 smear-negative specimens in which culture or pathology revealed M. tuberculosis. In addition, 2 smear-negative specimens and 8 smear-negative and culture-negative specimens were positive by PCR. These 10 samples were collected from the patients suspected as having tuberculosis by the clinical diagnosis based on the clinical history, characteristic radiographs, a positive PPD skin test and the effectiveness of anti-tuberculous drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on anabolic steroids. V. Sequential reduction of methandienone and structurally related steroid A-ring substituents in humans: gas chromatographic-mass spectrometric study of the corresponding urinary metabolites.

The biotransformation of methandienone (17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-one) in human adults, more particularly the sequential reduction of its A-ring substituents, was investigated by gas chromatography-mass spectrometry. Two pairs of 17-epimeric tetrahydro diols resulting from the stereoselective reduction of the delta 4- and 3-oxo groups and of the delta 1-function were characterized. The major diols were 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol, which were both excreted in the conjugate fraction in a 1:3.8 ratio. The immediate metabolic precursors of the 5 beta-diol, namely 17 beta-hydroxy-17 alpha-methyl-5 beta-androsta-1-en-3-one and 17 alpha-methyl-5 beta-androsta-1-en-3 alpha,17 beta diol and their corresponding 17-epimers, were also identified in post-administration urine samples. These data indicated that reduction of methandienone A-ring substituents proceeds according to the sequence. delta 4-, 3-oxo- and delta 1-. The A-ring reduction products of the structurally related steroids mestanolone, 17 alpha-methyltestosterone and oxymethone were also characterized and provided further analytical and metabolic evidence supporting the proposed route of methandienone A-ring reduction. It was also demonstrated using synthetic 17 beta-sulfate conjugates of methandienone and 17 alpha-methyltestosterone that their corresponding 17-epimers are formed by nucleophilic substitution by water of the labile sulfate moiety. The steroidal metabolites were identified on the basis of their characteristic mass spectral features and by comparison with authentic reference standards. Metabolic pathways accounting for the occurrence of the metabolites of interest in post-administration urine samples are proposed.

[The chromatographic-mass spectrometric analysis and detection of anabolic steroids in human urines and metabolic study].

Nineteen anabolic steroids were separately administered to healthy men. Anabolic steroid metabolites were isolated and extracted from the collected urine samples following an integrated procedure. The main metabolic pathways of these drugs were made clear after the investigation by GC-MS. Based on the obtained chromatographic-mass spectrometric data of anabolic steroids, a method for large scale and routine analysis of anabolic steroids was set up.

Studies on anabolic steroids. III. Detection and characterization of stanozolol urinary metabolites in humans by gas chromatography-mass spectrometry.

The metabolism of stanozolol (17 beta-hydroxy-17 alpha-methyl-5 alpha-androstano[3,2-c]pyrazole), an androgenic-anabolic steroid widely used in sport for the purpose of enhancing performance, was investigated in humans. The analysis method was based on the use of solid-phase extraction on the Sep-Pak C18 cartridge, enzymic hydrolysis of steroid conjugates and high-resolution gas chromatograph-mass spectrometric (GC-MS) analysis of trimethylsilylated steroid extracts. After administration of a single 20-mg oral dose, twelve metabolites including unchanged stanozolol were recovered predominantly from the conjugated steroid fraction and characterized by GC-MS. In the unconjugated fraction, 16 alpha-hydroxystanozolol, 17-epistanozolol, stanozolol and 3'-hydroxy-17-epistanozolol were the most abundant metabolites. In the aglycone fraction, 16 alpha- and 16 beta-hydroxystanozolol, stanozolol and 3'-hydroxystanozolol were the most abundant metabolites. Other metabolites resulted from regioselective hydroxylation of stanozolol at C-4, whereas other were 17-epimers of 3'- and 16 alpha-hydroxystanozolol. Further hydroxylation leading to the formation of four isomeric dihydroxylated metabolites was also observed. They were tentatively assigned the structures of 3',16 alpha-, 4 beta,16 alpha-, 3',16 beta- and 4 beta,16 beta-dihydroxystanozolol. The mass spectral features of their bis-N,O-trimethylsilyl derivatives obtained under electron-impact ionization are presented. The effect of pH on the relative recovery of some of these metabolites is also presented. The usefulness of this analytical methodology for the detection and identification of stanozolol urinary metabolites in doping-control situations is demonstrated. The metabolism of stanozolol is also discussed, and metabolic pathways accounting for the formation of its biotransformation products are proposed.

Studies on anabolic steroids. II--Gas chromatographic/mass spectrometric characterization of oxandrolone urinary metabolites in man.

The metabolism of 17 alpha-methyl-17 beta-hydroxy-2-oxa-5 alpha-androstan-3-one (oxandrolone) in man has been investigated by gas chromatography/mass spectrometry. After oral administration of a 10 mg dose to man, five metabolites were detected in the free fraction of the urinary samples. Oxandrolone, the major compound excreted in urine, was detected within 72 h after administration. During this period 35.8 and 8.4% of the administered dose was excreted as unchanged oxandrolone and 17-epioxandrolone, respectively. In addition, minute amounts of 16 alpha- and 16 beta-hydroxyoxandrolone and a delta-hydroxy acid resulting from the hydrolysis of the lactone group of oxandrolone were detected in the urine samples 8-60 h after administration. Furthermore, the susceptibility of oxandrolone to hydrolysis was investigated under several pH conditions. Extraction and fractionation of steroidal metabolites was achieved by using C18 and silica Sep Pak chromatography. The mass spectra of the metabolites are presented and major fragmentation pathways discussed.

[Determination of some androgens and anabolic steroids in human urine by HPLC].

A simple method for determining androgens and anabolic steroids in human urine by HPLC has been developed. The compounds studied are nandrolone, methandienone, testosterone, methyltestosterone, testosterone propionate and nandrolone phenylpropionate. The stationary phase used is C8 bonded silica. Isocratic elution was done with CH3OH-CH3CN-H2O (4:5:6) and programmed flow. Detection limit can be less than 1 ng at the wavelength of 254 nm. The standard curves for each steroid have been set using internal standard (progesterone) and peak height ratio. Linear relationship exists between the ratio and concentration for each steroid. High and stable recovery has been achieved using Sep-Pak C18 cartridges for urine sample clean-up. The operation of the method is simple. The enzymatic hydrolyzation of conjugated steroids in human urine has also been investigated.