Evidence against the Bm1P1 protein as a positive transcription factor for barbiturate-mediated induction of cytochrome P450BM-1 in bacillus megaterium.

Shaw GC, Sung CC, Liu CH, Lin CH.

Institute of Biochemistry, School of Life Science, National Yang-Ming University, Taipei 112, Taiwan, Republic of China.

The Bm1P1 protein was previously proposed to act as a positive transcription factor involved in barbiturate-mediated induction of cytochrome P450BM-1 in Bacillus megaterium. We now report that the bm1P1 gene encodes a protein of 217 amino acids, rather than the 98 amino acids as reported previously. In vitro gel shift assays indicate that the Bm1P1 protein did not interact with probes comprising the regulatory regions of the P450BM-1 gene. Moreover, disruption of the bm1P1 gene did not markedly affect barbiturate induction of P450BM-1 expression. A multicopy plasmid harboring only the P450BM-1 promoter region could increase expression of the chromosome-encoded P450BM-1. The level of expression is comparable with that shown by a multicopy plasmid harboring the P450BM-1 promoter region along with the bm1P1 gene. These results strongly suggest that the Bm1P1 protein is unlikely to act as a positive regulator for barbiturate induction of P450BM-1 expression. Finally, deletion of the Barbie box did not markedly diminish the effect of pentobarbital on expression of a reporter gene transcriptionally fused to the P450BM-1 promoter. This suggests that the Barbie box is unlikely to be a key element in barbiturate-mediated induction of P450BM-1.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9525898&dopt=Abstract barbiturate Butalbital Fioricet





Setting cutoff concentrations for immunoassay screening of postmortem blood.

Collison IB, Spiehler VR, Guluzian S, Sedgwick PR.

Forensic Science Services, Santa Ana, CA, USA.

The objective of this study was to establish the optimum immunoassay cutoff concentrations for screening postmortem blood from coroner's cases for drugs of abuse with a coated tube radioimmunoassay (RIA) to ensure that the results with the coated tube RIA would be equal to or better than those with the previously used double antibody RIA. Immunoassay results (positive or negative) blood were compared to confirmed results on those cases by GC/MS alone or in combination with GLC using either a NPD or FID detector. Four to seven potential cutoff concentrations were evaluated for the drug classes opiates, amphetamines, cocaine and metabolites, and barbiturates. Specimens were 350 postmortem blood specimens and liver homogenates. The cutoffs chosen for the coated tube RIA using this approach were 5 ng/mL morphine, 25 ng/mL methamphetamine, 500 ng/mL benzoylecgonine, and 500 ng/mL secobarbital. These cutoffs corresponded to a sensitivity and specificity of 94% and 96% for opiates, 93% and 86% for amphetamines, 91% and 96% for cocaine and metabolites and 91% and 87% for barbiturates. The double antibody RIAs were run on the same specimens with cutoffs of 20 ng/mL morphine, 50 ng/mL methamphetamine, 50 ng/mL benzoylecgonine and 1000 ng/mL phenobarbital. The sensitivity and specificity's for the double antibody immunoassay were: > 99% and 96% for opiates, 83% and 89% for amphetamines, 98% and 97% for cocaine, 79% and 95% for barbiturates.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9544549&dopt=Abstract barbiturate Butalbital Fioricet





A barbiturate screening assay for the Abbott AxSYM analyzer.

Adamczyk M, Douglas J, Grote J, Harrington CA.

Department of Chemistry, Abbott Laboratories, Abbott Park, Illinois 60064-3500, USA.

A fluorescence polarization immunoassay for barbiturates on the Abbott AxSYM analyzer is described. The assay displayed dilution linearity up to 1200 ng/mL; coefficients of variation varied from 5.96 to 8.61%; recovery varied from 94.9 to 105.3%; and sensitivity was less than 40 ng/mL. Good correlation between the standard six- and factory two-point calibration methods was observed. The Immunoassay demonstrated good cross-reactivity to several commonly prescribed barbiturates; low cross-reactivity with structurally similar compounds; low interference from endogenous substances, dyes, preservatives, and several commonly available adulterants; and good correlation with the TDx Barbiturate Urine assay.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9547406&dopt=Abstract barbiturate Butalbital Fioricet





Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography-mass spectrometry.

Namera A, Yashiki M, Okada K, Iwasaki Y, Ohtani M, Kojima T.

Department of Legal Medicine, Hiroshima University School of Medicine, Japan.

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography-mass spectrometry (GC-MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C18 disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol-water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform-isopropanol (3:1, v/v) from the cartridge. The eluate (1 microl) was injected into a GC-MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 microg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9551811&dopt=Abstract barbiturate Butalbital Fioricet