Shivering in patients recovering from CABG.
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Analgesics, Opioid↗
Biomedical subjects
L M Benson
Publications and source records attributed to L M Benson.
On-line HPLC-tandem mass spectrometry analysis of contaminants of L-tryptophan associated with the onset of the eosinophilia-myalgia syndrome.
The structural characterization of a number of contaminants of L-tryptophan (Trp) associated with eosinophilia myalgia syndrome has been performed for the first time by the powerful structural elucidation technique of tandem mass spectrometry coupled with on-line HPLC (LC-ESI-MS/MS). The identity of the contaminants: peaks UV-5, 3-(phenylamino)alanine, (PAA); E 1,1'-ethylidenebis(tryptophan); 200, 2-(3-indolylmethyl)-L-tryptophan; (all identified as case related) and peaks 1, 3-carboxy-1,2,3,4-tetrahydro-beta-carboline; 2, 3-carboxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline; 100, 2-(2,3 dihydroxy-1-[3-indolyl]propyl)-L-tryptophan; and 300 and 400, diastereomers of 3-carboxy-1-[3-indolyl-methyl]-1,2,3,4-tetrahydro-beta-carboline, have been confirmed by this technique. By comparison of tandem MS (MS/MS) data from these compounds with the MS/MS data of several other impurities, we have structurally characterized peaks CC, KK and OO, as well as two previously unreported components labeled as peak P18 and peak P31. Peak P18 was unresolved from the large Trp peak and has been characterized as indole-3-ethylamine. Peak P31 was previously unresolved from peak 200, a case related compound and therefore its structure is of extreme importance. This compound has been tentatively identified as 2-(3-indolyl)-L-tryptophan.
Chromatography, High Pressure Liquid↗
Recombinant Mycobacterium tuberculosis KatG(S315T) is a competent catalase-peroxidase with reduced activity toward isoniazid.
The presence of KatG(S315T), a mutation frequently detected in clinical isolates of Mycobacterium tuberculosis, has been associated with loss of catalase-peroxidase activity and resistance to isoniazid therapy. Wild-type KatG and KatG(S315T) were expressed in a heterologous host (Escherichia coli) and purified to homogeneity, and enzymatic activity was measured. The catalase activity for KatG(S315T) was reduced 6-fold, and its peroxidase activity was decreased <2-fold, compared with the activities for wild-type KatG. Pyridine hemochrome analysis demonstrated 1.1 +/- 0.1 hemes/subunit for wild-type KatG and 0.9 +/- 0.1 hemes/subunit for KatG(S315T), indicating that the difference in enzymatic activity is not the result of incomplete heme cofactor incorporation in KatG(S315T). High-performance liquid chromatography analysis showed that wild-type KatG was more efficient than KatG(S315T) at converting isoniazid to isonicotinic acid. These results demonstrate that KatG(S315T), as expressed in E. coli, is a competent catalase-peroxidase that exhibits a reduced ability to metabolize isoniazid.
Amino Acid Sequence↗
Application of capillary electrophoresis and related techniques to drug metabolism studies.
The use of capillary electrophoresis (CE) for the separation of small organic molecules such as pharmaceutical agents and drug/xenobiotic metabolites has become increasingly popular. This has arisen, at least in part, from the complimentary mode of separation afforded by CE when compared to the more mature technique of HPLC. Other qualities of CE include relative ease of method of development, rapid analysis, and low solvent consumption. The recent introduction of a variety of detector systems (including UV diode array, laser-induced fluorescence, conductivity) and the demonstrated coupling of CE to MS have also aided acceptance of this technology. In the present report, we review the role of CE coupled to various detector systems including a mass spectrometer for the characterization of both in vitro and in vivo derived drug metabolite mixtures. Attributes of CE for this application are demonstrated by discussion of metabolism studies of the neuroleptic agent haloperidol. Various aspects of the development and use of CE and CE-MS for the characterization of haloperidol metabolites, including criteria for selection of parameters such as pH, ionic strength, extent of organic modification, and the use of nonaqueous capillary zone electrophoresis are discussed. We also consider potential limitations of CE and CE-MS for drug metabolism research and describe the introduction of membrane preconcentration-CE (mPC-CE) and mPC-CE-MS as a solution that overcomes the rather poor concentration limits of detection of CE methods without compromising the resolution of analytes or separation efficiency of this technique.
Adult↗
Rapid loading of large sample volumes, analyte cleanup, and modified moving boundary transient isotachophoresis conditions for membrane preconcentration-capillary electrophoresis in small diameter capillaries.
Using a removable membrane preconcentration (mPC) cartridge, large sample volumes can be loaded prior to final assembly of the mPC capillary electrophoresis (CE) capillary. For narrow-bore (< or = 25 microns ID) uncoated mPC-CE capillaries, applied to peptide analysis, efficient moving boundary transient isotachphoresis (tITP) conditions at the onset of electrophoresis are described. The enhancement of mPC-CE-mass spectrometry (MS) technology afforded by rapid sample loading and modified moving boundary tITP conditions are demonstrated by analysis of major histocompatibility complex (MHC) class I peptides that were derived from a Kb precipitation of mouse EL-4 cells. Furthermore, we demonstrate the structural characterization of these immunologically significant molecules by mPC-CE-tandem mass spectrometry (mPC-CE-MS/MS).
Amino Acid Sequence↗
Preliminary investigations of preconcentration-capillary electrophoresis-mass spectrometry.
Analyte preconcentration on-line with capillary electrophoresis-mass spectrometry (PC-CE-MS) is described. Preconcentration cartridges were fabricated from PTFE tubing filled with ca. 1-2 mm bed of reversed-phase C18 HPLC packing or polymeric reversed-phase beads. The particle size of the stationary phase was of larger dimension than the internal diameter of the CE capillary. Therefore, PC-CE capillaries were assembled without frit material and held together by friction. The wide applicability of on-line PC-CE-MS is demonstrated by the analysis of solutions containing peptides, proteins, and synthetic analogues of putative metabolites of the neuroleptic agent haloperidol.
Amino Acid Sequence↗
Simultaneous structure-activity determination of disulfiram photolysis products by on-line continuous-flow liquid secondary ion mass spectrometry and enzyme inhibition assay.
Disulfiram (DSF) is used in the treatment of recovering alcoholics and exerts its effect by inhibiting the enzyme aldehyde dehydrogenase (ALDH). We analyzed a mixture of products derived photochemically from DSF with on-line microbore HPLC-continuous-flow liquid secondary ion mass spectrometry (HPLC-CF-LSI-MS). By utilizing the post-HPLC column split of solvent flow, a small proportion (ca. 5%) was sent directly into the mass spectrometer, and the remainder was collected. Simultaneous MS analysis and enzyme inhibition studies on ALDH were then possible. Furthermore, using HPLC-CF-LSI-MS-MS, we were able to structurally characterize an interesting sulfine compound that inhibited ALDH.
Aldehyde Dehydrogenase↗
Photolysis of sulfiram: a mechanism for its disulfiram-like reaction.
Sulfiram, a drug applied topically to treat scabies, produces effects similar to those of disulfiram after subsequent ingestion of ethanol. Disulfiram, used in aversion therapy in the treatment of alcoholism, inhibits hepatic aldehyde dehydrogenase (ALDH) causing an accumulation of acetaldehyde after ethanol ingestion. The increased tissue levels of acetaldehyde cause a spectrum of undesirable side-effects including flushing, nausea, vomiting, and tachycardia, which are referred to as the disulfiram reaction. Previous studies have shown that in vitro sulfiram is a very weak inhibitor of ALDH, but solutions of sulfiram markedly increase in potency with time. In the present study, fresh solutions of sulfiram were exposed to fluorescent room light under ambient conditions and analyzed at timed intervals by HPLC. At least eight products, including disulfiram, were formed in the light-exposed sulfiram solutions, but not in solutions kept in the dark. Structural characterization of two of the photolysis products was obtained by on-line microbore HPLC-mass spectrometry (mu LC-MS) and on-line microbore HPLC-tandem mass spectrometry (mu LC-MS/MS) using continuous flow-liquid secondary ion mass spectrometry (CF-LSIMS) as the primary ionization method. Sulfiram was converted to disulfiram at an initial rate of 0.7%/hr, and the formation of disulfiram correlated with the increase in ALDH inhibition in vitro. The results of this investigation show that while sulfiram is a weak inhibitor of ALDH in vitro, it is readily photoconverted to disulfiram, a very potent inhibitor of ALDH, which may explain the adverse reaction to ethanol after sulfiram therapy.
Aldehyde Dehydrogenase↗
Investigation of the in vitro metabolism of the H2-antagonist mifentidine by on-line capillary electrophoresis-mass spectrometry using non-aqueous separation conditions.
The in vitro metabolism of mifentidine, a prototype second-generation histamine H2-antagonist, is investigated using on-line capillary electrophoresis-mass spectrometry (CE-MS) by analysis of hepatic microsomal incubates. Consideration of the hydrophobicity of this drug and putative metabolites led to the development of a non-aqueous CE separation medium consisting of 5 mM NH4OAc in methanol containing 100 mM acetic acid. Benefits of non-aqueous media in CE-MS studies of small hydrophobic molecules are discussed. In addition, we elucidate both chemical transformations and the in vitro metabolism of mifentidine using guinea pig hepatic microsomes.
Animals↗
Investigation of drug metabolism using capillary electrophoresis with photodiode array detection and online mass spectrometry equipped with an array detector.
The application of capillary electrophoresis (CE) with photodiode array detection (DAD) and on-line CE-mass spectrometry (CE-MS) equipped with a position and time resolved (PATRIC) focal plane detector for analysis of both in vitro and in vivo drug metabolism is demonstrated. Separation of metabolites derived from the neuroleptic drug haloperidol, by CE, using a simple, volatile run buffer containing 50 mM ammonium acetate with 10% methanol and 1% acetic acid is reported. The potential utility of CE-DAD for screening drug metabolite mixtures derived from hepatic microsomal incubations is demonstrated for haloperidol (HAL). Also the potential problems associated with using this technology to screen human urine samples for HAL metabolites is discussed. Furthermore, the usefulness of CE-MS and CE-electrospray ionization skimmer collision induced dissociation-MS (CE-ESI-CID-MS) in identification and structure elucidation of HAL metabolites derived from both a guinea pig hepatic microsomal incubation and urine from a patient treated with 0.5 mg/day of HAL is shown. The utility of such an approach in the general area of clinical pharmacology is also discussed.
Animals↗
Investigation of the metabolic fate of the neuroleptic drug haloperidol by capillary electrophoresis-electrospray ionization mass spectrometry.
Combined capillary electrophoresis-mass spectrometry (CE-MS) is a powerful analytical tool for the characterization of components of complex mixtures. Furthermore, when interfaced with an electrospray ionization (ESI) source, skimmer induced fragmentation can yield structural information that aids compound identification. We show that these techniques can be used to probe the metabolic fate of drugs by investigation of the phase I metabolism of the widely used neuroleptic drug haloperidol. In addition, we demonstrate differences of in vitro metabolism of haloperidol by mouse and guinea pig hepatic microsomes.
Animals↗
Investigation of the metabolism of the neuroleptic drug haloperidol by capillary electrophoresis.
Free solution capillary electrophoresis (FSCE) conditions were previously reported to be of limited use for the separation of pharmaceuticals, since many of these compounds are neutral. We show that by consideration of compound hydrophobicity and ionisable functional groups, FSCE conditions can be developed to effect the separation of a drug and its phase I metabolites. This is brought about by adding a suitable organic modifier to aid solubility, and modifying pH to effect a change in the mass to charge ratio of the metabolites present. Furthermore, we show that in this drug metabolism study, FSCE presents an advantage over both reversed-phase HPLC and micellar electrokinetic chromatography. We also demonstrate the use of FSCE for investigation of the phase I metabolites produced by the in vitro incubation of haloperidol (a neuroleptic agent) with both mouse and guinea pig hepatic microsomes and show that such an approach can be used to detect both qualitative and quantitative differences in species metabolism.
Animals↗
Murine pharmacokinetics and metabolism of penclomedine [3,5-dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine, NSC 338720].
Penclomedine, a highly substituted pyridine derivative, has been selected by the National Cancer Institute for evaluation as a potential anticancer agent based on antitumor activity observed in murine tumor models following i.v., p.o., and i.p. administration. We have developed a reverse-phase high performance liquid chromatography assay for PEN, and subsequently investigated murine pharmacokinetics and metabolism. Following rapid i.v. injection of PEN (300 mg/m2) to mice, plasma elimination was best described by a 2-compartment open model with an elimination phase half-life, total body clearance, and steady-state distribution volume of 69 min, 114 ml/min/m2, and 4800 ml/m2, respectively. While PEN displayed good p.o. absorption, bioavailability of PEN after p.o. administration was approximately 2% of that observed following i.v. administration. Metabolism contributed substantially to drug clearance, and total metabolites were slowly eliminated from plasma. After i.v. and p.o. administration of radiolabeled PEN, less than 0.2% of the parent drug was excreted in the 48-h urine, and 25-30% of the total radioactivity was recovered in urine. NADPH-dependent oxidative and reductive metabolism was observed when penclomedine was incubated with mouse microsomal preparations. Microsomal reductive metabolism of PEN led to formation of a metabolite tentatively identified as a molecule formed by dimerization of the radical species produced by cleavage of chlorine from the trichloromethyl moiety of penclomedine.
Animals↗
Quantitation of endogenous substance P by on-line microcolumn liquid chromatography/continuous-flow fast-atom bombardment mass spectrometry.
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Animals↗
Pig skin as test substrate for evaluating topical antimicrobial activity.
The feasibility of using pathogen-contaminated pig skin as a model substrate for evaluating skin disinfectants was demonstrated. A test methodology is described that is safe, convenient to use, and adaptable to a variety of hand-washing conditions. The treatment protocol, pathogen contamination conditions, and application technique variables can all be carefully controlled to simulate clinical use conditions. The number of organisms transferred by contact was compared with the total organism count on the pig skin. The quantity of organisms transferred ranged from 10 to 60% of the total organisms, depending on the nature of the contamination conditions. The cumulative results of multiple imprint and stripping measurements were consistent with the concentration of inoculated organisms. Tests with alcohol solutions validated the methodology and clearly showed the dependence of topical antimicrobial activity on both the concentration and structure of the alcohol. Activity increased with increasing alcohol concentration and in the following order: ethanol, isopropanol, and n-propanol. All of the alcohols became less active as the severity of the test conditions was increased, i.e., higher inoculum levels for a longer incubation time before treatment. The contact imprint and stripping methods used to evaluate bacterial growth on the skin clearly showed that the alcohol treatments reduced but did not eliminate the inoculated pathogens. It was found that long lifetimes (several hours) for pathogens on the skin are possible under some environmental conditions. This observation strongly suggests that frequent hand washing is a necessary infection control practice even when opportunities for repeated pathogen contamination have not occurred.
1-Propanol↗
Influence of hepatic oxidative metabolism on the immunosuppressive activities of 6-thiopurines.
It has been suggested that in vitro and in vivo hepatic metabolism of 6-thiopurine and its prodrug azathioprine generates immunosuppressive metabolites with far greater activity than the parent drugs. To examine this possibility, in vitro and in vivo drug-metabolizing systems were interfaced with in vitro immune response assays for the detection of active 6-thiopurine metabolites. In this way, it was demonstrated that preincubation of 6-thiopurine with hepatic microsomes from Balb/c mice does not enhance the immunosuppressive activity of the parent drug in in vitro lymphocyte proliferation assays performed with Balb/c splenocytes stimulated by T and B cell mitogenic lectins. In fact, prior microsomal metabolism generally decreased the inhibitory effects of 6-thiopurine on lymphocyte mitogenic responses, indicating that any metabolites formed under these conditions were relatively inactive. In contrast, the immunosuppressive effects of cyclophosphamide, which was used as a positive control in this system, were profoundly increased by microsomal metabolism. It was shown, moreover, that in vivo metabolism of 6-thiopurine and azathiopurine did not lead to the generation of metabolites with detectably greater immunosuppressive activity than the parent drugs. The circulating immunosuppressive activity present in the serum of drug-treated Balb/c mice, when quantitated in vitro with a sensitive murine mixed lymphocyte culture system, was found to correlate strongly with the serum levels of the unmetabolized thiopurines, which were measured specifically by high-pressure liquid chromatography assay. Taken together, these results fail to support earlier hypotheses that blood-borne active metabolites contribute significantly to the immunosuppressive actions of 6-thiopurines.
Animals↗
Synthesis and pH-dependent stability of purine-6-sulfenic acid, a putative reactive metabolite of 6-thiopurine.
Previous studies have shown that 6-thiopurine is metabolically activated by hepatic cytochrome P-450 to an intermediate capable of binding to proteins by a mixed disulfide linkage. The identity of the active metabolite was postulated to be purine-6-sulfenic acid. In the present report, we describe the synthesis of the sulfenic acid derivatives of 6-thiopurine and two structurally similar compounds, 9-methyl-6-thiopurine and 4-mercapto-1H-pyrazolo[3,4-d]-pyrimidine. The unusual pH-dependent stability profiles of these compounds in buffered aqueous media are presented and explained on the basis of a disproportionation mechanism of sulfenic acid decomposition. Studies with radiolabeled purine-6-sulfenic acid demonstrate that this species binds directly to hepatic microsomal protein. These results support the proposed involvement of purine-6-sulfenic acid in the metabolic activation and tissue binding of 6-thiopurine.
Animals↗
Investigation of the histocompatibility of the NYA:NYLAR mouse colony by skin grafting.
The histocompatibility status of the Nya:NYLAR mouse colony was studied by exchange of skin grafts between female mice. The colony had been divided into two portions since 1962, a larger, outbred stock (Nya:NYLAR), and a smaller, inbred strain (NYLR/Nya). The results of skin graft exchanges between mice of the inbred strain indicated that they were skin-compatible. There was weak skin-incompatibility within the outbred stock and between this stock and the inbred strain, and strong skin-incompatibility between the outbred stock and outbred Webster Swiss mice.
Animals↗