Alteration of transit time and direction of flow to probe the heterogeneous distribution of conjugating activities for harmol in the perfused rat liver preparation.

Previous studies in the once-through perfused rat liver preparation have shown that the techniques of normal and retrograde delivery of substrate and computer simulation of enzyme distributions along the sinusoidal flow path in liver were useful in delineating the relative distributions of sulfation and glucuronidation activities for harmol metabolism (Pang et al., J. Pharmacol. Exp. Ther. 224: 647-653, 1983). The observed steady-state hepatic extraction ratios of harmol and the steady-state formation rates of harmol sulfate and harmol glucuronide were consistent with three enzyme-distribution models which described a proximal localization of sulfation activity and a more distal distribution of glucuronidation activities. The present study was a further refinement on the definition of the distribution of these activities. The experimental approach used included the perturbation of delivery of harmol (10 microM) at 8, 12 and 16 ml/min by normal flow, and the alternation of normal and retrograde directional flows for delivery of harmol (10 microM) at constant hepatic blood flows (8, 12 or 16 ml/min) in the single-pass perfused rat liver preparation. The observed steady-state sulfation and glucuronidation rates were compared against predicted values afforded by models previously shown to be adequate and additional enzyme-distributed models. The observed and predicted data point to a periportal distribution of sulfation activity and an even distribution of glucuronidation activity for the metabolism of harmol.

An understanding of the role of enzyme localization of the liver on metabolite kinetics: a computer simulation.

The metabolic sequence of drug, D, to its primary (MI) and terminal (MII) metabolites as mediated by enzymes A and B, respectively, was chosen to illustrate metabolizing activities among hepatocytes in different regions of the liver lobule. Six models of distributions of the hepatocellular activities (intrinsic clearances for A and B) were defined with respect to the flow path in liver, and the concentrations D, MI, and MII in the liver were simulated. The extent of sequential metabolism of the primary metabolite was compared for these six models of enzymic distributions. It was found that when the average hepatic intrinsic clearances of A and B were high (almost complete extraction of both drug and primary metabolite during their single passage through the liver), the distributions of A and B were not important determinants of metabolite kinetics. By contrast, when the average hepatic intrinsic clearances of A and B were both low, the distributions of A and B exerted profound effects on metabolite kinetics. The sensitivity to enzymic distribution in this region, however, was difficult to assess due to difficulties in detecting low levels of MI and MII. The effects of enzymic distributions on metabolite disposition would be better detected in compounds (drug and metabolite) with intermediate extraction ratios.

Normal and retrograde perfusion to probe the zonal distribution of sulfation and glucuronidation activities of harmol in the perfused rat liver preparation.

The role of zonal distributions of metabolic activities (sulfation and glucuronidation) in the liver on the kinetics of harmol conjugation was investigated. A computer simulation approach was adopted to better understand the effects of distributions of these conjugation activities in competition for a common drug substrate. Several distributions of the sulfation and glucuronidation systems were defined with respect to the flow path of blood; the conjugation activities along the flow path were in turn translated as time elapsed after entry of the substrate via blood into the organ. Realistic values of Km and Vmax for the sulfation and glucuronidation systems were assigned in the simulations. Directional flow, namely, normal vs. retrograde delivery of substrate was used as an additional variable. When the "center" of distribution of the sulfation system was anterior to that for glucuronidation, the steady-state hepatic extraction ratio of harmol (E) would increase, whereas the ratio of the steady-state rates of formation of harmol sulfate to harmol glucuronide (S/G ratio) would decrease when harmol was presented in a reversed direction (via retrograde perfusion), as compared with normal directional flow to the liver. The converse was true for an anterior distribution of the glucuronidation system; E would decrease, whereas S/G would increase with retrograde perfusion. To evaluate such zonal differences experimentally, perfusion studies were conducted in livers of male Wistar rats. A constant concentration of harmol (50 microM) was delivered under constant hepatic flow rate (10 ml/min/liver) by normal and retrograde perfusions to the same rat liver preparation. The steady-state hepatic E was higher (P less than .005) during retrograde perfusion than during normal perfusion, whereas the S/G ratio was significantly decreased (P less than .0005). The observations suggest an intercellular difference in the distribution of the two conjugating systems and are consistent with the view of the center of distribution for the sulfation system being anterior to the center of distribution for the glucuronidation system along the normal flow path of blood in the liver.

Children absorb tris-BP flame retardant from sleepwear: urine contains the mutagenic metabolite, 2,3-dibromopropanol.

The flame retardant, tris(2,3-dibromopropyl)phosphate (tris-BP), which is a mutagen and causes cancer and sterility in animals is absorbed from fabric by people. 2,3-Dibromopropanol, a metboloite of tris-BP and a mutagen itself, has been found in the urine samples of ten children who were wearing or who had worn tris-BP-treated sleepwear. Eight of these children were wearing well-washed sleepwear and the possibility of absorption of tris-BP from well-washed sleepwear discussed. 2,3-Dibromopropanol was not found in the urines of one child and one adult who had never worn tris-BP-treated garments.

Development and use of analytical systems based on mass spectrometry.

Contemporary analytical systems based on mass spectrometry include as components a gas chromatograph, a mass spectrometer, and a computer. The form of operation is usually in electron impact ionization mode for identification and structural studies, and in chemical ionization mode for quantitative analyses. Important stages in the development of these systems included the design of "molecule separators" for the concentration of solutes in the gas phase, the use of mass spectrometers as specific ion detectors, the introduction of chemical ionization techniques, and the development of computer-based operation, data acquisition, and data analysis capabilities. A current line of investigation is concerned with the design and use of systems based on atmospheric pressure ionization. Samples are ionized in a small reaction chamber external to the low-pressure region of a quadrupole mass analyzer. The primary source of electrons is a 63Ni foil or a corona discharge. The ionization process leading to positive ions involves a sequence of ion molecule reactions, usually electrons leads to carrier gas ions leads to reagent ions leads to sample component ions. Negative ions may be formed by direct electron attachment, or by ion molecule reactions that include new types of elimination reactions. The source will accept a variety of gases and solvents. The sample may be introduced in the gas phase without solvents, by probe injection, or in the effluent stream from a gas chromatograph. Samples may be introduced in the liquid phase in solvents by injection after the fashion of gas chromatography or in the effluent stream from a high-performance liquid chromatograph. The novel aspects of atmospheric pressure ionization mass spectrometry lie in its versatility and high sensitivity of detection. Few clinical chemistry laboratories now use these systems. Significant future uses are likely to be in analytical work involving therapeutic drug monitoring and studies of drug metabolism, and in analyses for environmental biohazards including pesticides, herbicides, polyhalobiphenyls, dibenzodioxins, and other toxic compounds.

Gas chromatographic-mass spectrometric-computer methods in the study of drug metabolism.

The computer-assisted interpretation of repetitively scanned mass spectra can be of great value in the analysis of a complex mixture even in the absence of a library of reference spectra. We have written a set of interpretation programs for a PDP11/45 laboratory computer. A file structure is used that combines economy of storage with ease of creation and retrieval. User interaction is by a simple command language implemented by an interpreter. The program structure is such that additional functions can be added easily.

The use of gas chromatographic-mass spectrometric-computer systems in pharmacokinetic studies.

Pharmacokinetic studies involving plasma, urine, breast milk, saliva and liver homogenates have been carried out by selective ion detection with a gas chromatographic-mass spectrometric-computer system operated in the chemical ionization mode. Stable isotope labeled drugs were used as internal standards for quantification. The half-lives, the concentration at zero time, the slope (regression coefficient), the maximum velocity of the reaction and the apparent Michaelis constant of the reaction were determined by regression analysis, and also by graphic means.

Analysis of nanogram quantities of norethisterone in plasma using a GC-MS-COM selective ion detection procedure.

PIP: A procedure to detect and quantify nanogram amounts of the contraceptive steroid norethisterone (17alpha-ethinyl-17beta-hydroxy-4-estren-3-one) in human plasma is described. Norgestrel (racemic 13beta-ethyl-17alpha-ethinyl-17beta-hydroxy-4-gonen-3-one) was added to the plasma as an internal reference compound. Norethisterone and norgestrel were isolated from the plasma using a benzene and ammonium carbonate extraction procedure and were treated with trimethylsilylimidazole and potassium acetate to form TMS-enol TMS derivatives. Analyses of norethisterone and norgestrel were done by selective ion detection of the molecular ions using a combination of gas chromatography, mass spectrometry, and a computer.^ieng