Mechanism-based inactivation of cytochrome P450 2B1 by N-benzyl-1-aminobenzotriazole.

The kinetics of inactivation of cytochrome P450 2B1, the major phenobarbital inducible rat hepatic P450, by N-benzyl-1-aminobenzotriazole (BBT) were characterized. Purified, reconstituted P450 2B1 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylase activity was inhibited by BBT in a mechanism-based manner. The loss of O-deethylase activity followed pseudo-first-order kinetics and was NADPH and BBT dependent. After a 5 min incubation, greater than 90% of the 2B1 activity was lost, whereas more than 70% of the ability of the reduced enzyme to bind CO was maintained. Inclusion of 10 mM glutathione in the inactivation reaction lowered the rate of inactivation (k(inactivation)) and increased the partition ratio without significantly affecting the inactivator concentration required for half-maximal inactivation (K(I)). The maximal rate constant for inactivation at 23 degrees C was 0.24 min(-1) without and 0.15 min(-1) with glutathione. The apparent K(I) was 2 microM in both cases. The extrapolated partition ratios were 4 and 9 without and with 10 mM glutathione, respectively. Consistent with mechanism-based inactivation, the loss of 7-EFC O-deethylase activity was irreversible, was not due to product inhibition, was saturable, and could be slowed by including increasing concentrations of competing substrate. However, the inactivated P450 2B1 was still able to metabolize substrate if iodosobenzene was used as an alternate oxidant. Inactivation of 2B1 with either N-[14C]-7-benzyl-1-aminobenzotriazole (BBT) or N-benzyl-1-amino-[14C]-2,3-benzotriazole resulted in the incorporation of covalent radiolabel into the apoprotein. The stoichiometry of labeled metabolite adduct to protein was approximately 0.4:1 in both cases. Identification of metabolites revealed the formation of 1-aminobenzotriazole, benzotriazole, benzaldehyde, and a new metabolite (27) during catalysis of BBT by P450 2B1. Together, these data suggest that P450 2B1 could be inactivated and labeled by more than one metabolite.

Dirofilaria immitis: do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta?

Endothelial cells modulate the function of their underlying smooth muscle. Thus, altered endothelial behavior could be important in the pathogenesis of vascular and lymphatic diseases, including human and animal filariasis. Endothelium-dependent relaxation is depressed in both in vivo canine femoral artery of dogs infected with Dirofilaria immitis and in vitro rat aorta exposed to adult D. immitis. The experiments reported here were designed to determine if filarial cyclooxygenase products could depress endothelium-dependent relaxation in vitro. Pretreatment of the parasites, but not the vascular ring, with either indomethacin or aspirin, prevented filarial-induced depression of relaxation. Analysis of heartworm-conditioned medium by gas chromatography--mass spectrometry revealed two peaks in the biologically active medium that were not present in the control. One peak had a retention time and chromatographic profile characteristic of derivatized PGD2 standard, and the other was not identified. Incubation of the vascular ring with PGD2 mimicked filarial-induced depression of endothelium-dependent relaxation at low, but not high, concentrations of acetylcholine. Thus, filarial PGD2 may be involved in altered endothelium-dependent relaxation seen in heartworm-infected dogs.

Complex mixture analysis based on gas chromatography-mass spectrometry with time array detection using a beam deflection time-of-flight mass spectrometer.

A beam deflection time-of-flight mass spectrometer was developed in conjunction with an integrating transient recorder to provide time array detection, permitting high mass spectral scan file acquisition rates for complex mixture analysis by capillary gas chromatography-mass spectrometry (GC-MS). Results are presented for the analysis of a urinary organic acid mixture by GC-MS at a scan file acquisition rate of 10 scan files per second (sf/s), showing the advantages of such data collection in the deconvolution of partially resolved components. The reconstructed total ion current (RTIC) chromatogram available from data acquired at this scan file generation rate is shown to be comparable to the profile obtained from a flame ionization detector in representing the chromatography performed under identical experimental parameters. The RTIC chromatogram available from the database obtained at 10 sf/s is compared with that available from a database obtained at 1 sf/s, the latter representing that scan rate typically used with most GC-MS instruments. The advantages of the higher scan file acquisition rate in representing the chromatographic profile and in allowing mass spectral data to be obtained for components in the complex mixture that are unresolved chromatographically are discussed.

Interactive and multi-sensory analysis of complex mixtures by an automated gas chromatography system.

Methods are presented for increased human-computer interactions in the analysis of metabolic profiles of urinary organic acids. Complex mixtures of trimethylsilylated derivatives are separated by capillary gas-liquid chromatography on a 25-m bonded-phase, fused-silica column. Retention times and areas, determined by a printing integrator, are transferred to a personal computer along with the digitized output of the flame-ionization detector. Post-run computer analysis gives enhanced visual graphic displays with software-driven zooming, scrolling, peak identification and quantitation. In addition, auditory (musical) forms can be produced that increase the scope of human interaction. This novel approach catalyzes simultaneous use of computer calculations and human intuition, optimizing their combined cognitive powers for accurate qualitative and quantitative analyses of these complex mixtures.

Metabolic profiles of urinary organic acids recovered from absorbent filter paper.

We evaluated for reliability and reproducibility a semiquantitative gas-chromatographic assay of organic acids in samples of normal urine recovered from absorbent filter paper. We also evaluated this method for use in diagnosis of some of the more common organic acidurias. Transfer of urine from diapers to absorbent filter paper eases the usual trauma of specimen collection from young children; it also simplifies sample storage and shipment.