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D W Potter

Publications and source records attributed to D W Potter.

33 records · Page 2Linked to original sources
PubMed1987

Direct analysis of rat bile for acetaminophen and two of its conjugated metabolites via thermospray liquid chromatography/mass spectrometry.

Bile from rats treated with acetaminophen was analyzed by direct injection onto a thermospray liquid chromatography/mass spectrometry (LC/MS) system. Two conjugated metabolites of acetaminophen were separated by the high-pressure liquid chromatographic system and analyzed by mass spectrometry. The conjugates were identified as the glutathione-acetaminophen conjugate and the glucuronide-acetaminophen conjugate by comparison of the chromatographic retention times and the mass spectra to that of the synthetic standards. No evidence of acetaminophen metabolites was observed when bile samples were subjected to direct analysis by fast atom bombardment mass spectrometry.

Acetaminophen↗
PubMed1987

A sensitive immunochemical assay for acetaminophen-protein adducts.

The hepatotoxicity of acetaminophen may be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine which binds covalently to protein primarily as 3-(cystein-S-yl)acetaminophen. We have developed an avidin biotin-amplified competitive enzyme-linked immunosorbent assay to detect protein-bound acetaminophen. This assay utilizes antisera from rabbits immunized with 3-(N-acetyl-L-cystein-S-yl)acetaminophen coupled via the carboxyl group to primary amino groups on the protein keyhole-limpet hemocyanin. The competitive enzyme-linked immunosorbent assay utilizes metallothionein derivatized with N-acetyl-p-benzoquinone imine (acetaminophen-bound metallothionein) and quantitation was obtained by competition of acetaminophen-derivatives for a limited amount of antibody in the presence of excess solid phase acetaminophen-bound metallothionein. Synthetic 3-(N-acetyl-L-cystein-S-yl)acetaminophen, acetaminophen bound to mouse 9,000 X g supernatant, 100,000 X g supernatant, microsomes, as well as acetaminophen-bound metallothionein were inhibitory. The 50% inhibition for 3-(N-acetyl-L-cystein-S-yl)acetaminophen was 110 fmol/well. In contrast, free acetaminophen was 6200 times less efficient as an inhibitor. The mean 50% inhibition for protein-bound acetaminophen was 2.89 pmol/well. The utility of the method to detect acetaminophen-protein adducts in biological samples was confirmed by detection of NADPH-dependent binding of acetaminophen to microsomal proteins.

Acetaminophen↗
PubMed1986

Aerobic and anaerobic reduction of nitrated pyrenes in vitro.

Nitrated pyrenes are mutagenic and tumorigenic environmental pollutants that are activated to DNA-binding derivatives via nitroreduction. We have investigated the enzymatic nitroreduction of 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene to determine if differences in the extent of nitroreduction may help explain differences in their biological potencies. Each nitrated pyrene was incubated aerobically and anaerobically with 105,000 X g supernatant (S105) from Salmonella typhimurium TA98 and the nitroreductase-deficient strain, TA98NR, and with cytosol and microsomes from rat and human liver. Under anaerobic conditions, 1-nitropyrene and 1,3-dinitropyrene were reduced by TA98 S105 to a lesser extent than 1,6- and 1,8-dinitropyrene. The extent of 1,6- and 1,8-dinitropyrene metabolism was not altered relative to TA98 when using TA98NR S105, but the nitroreduction of 1-nitropyrene and 1,3-dinitropyrene was decreased. Both rat and human liver cytosol and microsomes reduced 1,6- and 1,8-dinitropyrene to greater extents than 1-nitropyrene and 1,3-dinitropyrene. Under aerobic conditions rat and human liver cytosols were similar to TA98 S105 in that aminopyrene decreased while nitrosopyrene formation increased. By comparison, oxygen decreased the microsomal formation of both nitrosopyrenes and aminopyrenes. The reduction of succinoylated cytochrome c was measured during the hepatic metabolism of nitro- and nitrosopyrenes under aerobic conditions. The data indicated that reduced nitro- and nitrosopyrene intermediates were directly reducing succinoylated cytochrome c and that the assay could be used as a measure of aerobic nitroreduction. These studies demonstrate that 1,6- and 1,8-dinitropyrene are reduced to a greater extent than 1-nitropyrene and 1,3-dinitropyrene, which corresponds to their relative biological potencies as mutagens and carcinogens. Furthermore, although more extensive nitroreduction is detected under anaerobic conditions, the nitroreduction that occurs aerobically may be important for the mutagenic and tumorigenic properties of these compounds.

Animals↗
PubMed1986

Reactions of N-acetyl-p-benzoquinone imine with reduced glutathione, acetaminophen, and NADPH.

Synthetic N-acetyl-p-benzoquinone imine reacted with reduced glutathione (GSH), [14C]acetaminophen, and NADPH. It reacted rapidly with GSH to yield acetaminophen (33%) and 3-(glutathion-S-yl)acetaminophen (67%), and with acetaminophen or NADPH to yield acetaminophen polymers. The data suggested that N-acetyl-p-benzoquinone imine was reduced by GSH to form acetaminophen but primarily reacted with GSH to form 3-(glutathion-S-yl)acetaminophen. The evidence further suggested that N-acetyl-p-benzoquinone imine comproportionated with [14C]acetaminophen to yield a mixture of radioactive and nonradioactive N-acetyl-p-benzosemiquinone imine which subsequently formed acetaminophen polymers by a radical coupling reaction. [14C]Acetaminophen was incorporated into the acetaminophen polymers. The amount of 14C incorporation was dependent on the initial concentration of [14C]acetaminophen and N-acetyl-p-benzoquinone imine. An increase in the ratio of [14C]acetaminophen to N-acetyl-p-benzoquinone imine resulted in an increase in [14C] acetaminophen incorporation into the acetaminophen polymers. NADPH reduced N-acetyl-p-benzoquinone imine to acetaminophen and acetaminophen polymers were formed. When [14C]N-acetyl-p-benzoquinone imine was incubated without acetaminophen, only minor amounts of acetaminophen polymerization were observed.

Acetaminophen↗
PubMed1986

Horseradish peroxidase-catalyzed oxidation of acetaminophen to intermediates that form polymers or conjugate with glutathione.

Horseradish peroxidase catalyzed the polymerization of acetaminophen. Addition of reduced glutathione (GSH) to reaction mixtures resulted in decreased polymerization and formation of minor amounts of GSH-acetaminophen conjugates. The conjugates were identified as 3-(glutathion-S-yl)acetaminophen and 3-(glutathion-S-yl)diacetaminophen. Horseradish peroxidase also catalyzed polymerization of synthetic 3-(glutathion-S-yl)acetaminophen to a dimer conjugate. In contrast to acetaminophen, 3-(glutathion-S-yl)acetaminophen oxidation was slowly catalyzed by horseradish peroxidase. However, in reaction mixtures containing equimolar concentrations of acetaminophen and synthetic 3-(glutathion-S-yl)acetaminophen, the formation of 3-(glutathion-S-yl)diacetaminophen and 3-(diglutathion-S-yl)diacetaminophen was rapid and accounted for approximately 95% of the products, whereas acetaminophen polymers accounted for only 5% of the products. These findings suggest that horseradish peroxidase catalyzed the one-electron oxidation of acetaminophen to N-acetyl-p-benzosemiquinone imine which preferentially polymerized rather than reacted with GSH. N-Acetyl-p-benzosemiquinone imine may also oxidize 3-(glutathion-S-yl)acetaminophen to form acetaminophen and 3-(glutathion-S-yl)-N-acetyl-p-benzosemiquinone imine. The data indicate that once this conjugate radical is formed it reacts with either N-acetyl-p-benzosemiquinone minine or 3-(glutathion-S-yl)-N-acetyl-p-benzosemiquinone imine via a radical termination mechanism.

Acetaminophen↗
PubMed1985

Identification of acetaminophen polymerization products catalyzed by horseradish peroxidase.

Horseradish peroxidase catalyzed the H2O2-dependent oxidation and polymerization of acetaminophen. Six acetaminophen polymers were isolated from horseradish peroxidase reaction mixtures by semipreparative high pressure liquid chromatography. Chemical structures were determined by a combination of electron impact and chemical ionization mass spectrometry and 500-MHz proton magnetic resonance spectroscopy. Two dimers, three trimers, and one tetramer were identified. The polymers formed primarily through a covalent bond between carbons ortho to the hydroxyl group, and to a lesser extent, between the carbon ortho to the hydroxyl group and the amino group of another acetaminophen molecule. Greater than 99% of the polymerization reaction products were quenched by the addition of 2.0 mM ascorbate. High acetaminophen concentration favored dimer formation, whereas low acetaminophen concentration favored formation of trimers and tetramers. Since approximately 1 mol of H2O2 was consumed per mol of covalent ligand formed between acetaminophen molecules, these products probably result from free radical termination reactions.

Acetaminophen↗
PubMed1985

Mechanism of microsomal metabolism of benzene to phenol.

The mechanism of microsomal hydroxylation of benzene to phenol has been studied by examining the microsomal metabolism of the specifically deuterated derivative 1,3,5-[2H3]benzene. Evidence for the formation of the following four products was obtained: 2,3,5-[2H3]phenol, 3,5-[2H2]phenol, 2,4,6-[2H3]phenol, and 2,4-[2H2]phenol. The presence of 2,3,5-[2H3]phenol and 2,4-[2H2]phenol shows that, in the microsomal metabolism of benzene to phenol, a NIH shift had occurred. A deuterium isotope effect (kH/kD) of approximately 4 was detected in both the meta- and para-deuterated phenols. This finding indicates that cyclohexadienone, formed either by isomerization of the epoxide or directly from the enzyme-substrate complex, is a major intermediate in the metabolism of benzene to phenol.

Animals↗
PubMed1984

Stereoselective monooxygenation of carcinostatic 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by purified cytochrome P-450 isozymes.

Three highly purified forms of liver microsomal cytochrome P-450 (P-450a, P-450b and P-450c) from Aroclor 1254-treated rats catalyzed 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) monooxygenation in the presence of purified NADPH-cytochrome P-450 reductase, NADPH, and lipid. Differences in the regioselectivity of CCNU and MeCCNU monohydroxylation reactions by the cytochrome P-450 isozymes were observed. Cytochrome P-450-dependent monooxygenation of CCNU gave only alicyclic hydroxylation products, but monooxygenation of MeCCNU gave alicyclic hydroxylation products, an alpha-hydroxylation product on the 2-chloroethyl moiety, and a trans-4-hydroxymethyl product. A high degree of stereoselectivity for hydroxylation of CCNU and MeCCNU at the cis-4 position of the cyclohexyl ring was demonstrated. All three cytochrome P-450 isozymes were stereoselective in primarily forming the metabolite cis-4-hydroxy-trans-4-Methyl-CCNU from MeCCNU. The principal metabolite of CCNU which resulted from cytochromes P-450a and P-450b catalysis was cis-4-hydroxy CCNU, whereas the principal metabolites from cytochrome P-450c catalysis were the trans-3-hydroxy and the cis-4-hydroxy isomers. Total amounts of CCNU and MeCCNU hydroxylation with cytochrome P-450b were twice that with hepatic microsomes from Aroclor 1254-treated rats. Catalysis with cytochromes P-450a and P-450c was substantially less effective than that observed with either cytochrome P-450b or hepatic microsomes from Aroclor 1254-treated rats.

Animals↗
PubMed1983

Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase.

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.

Anaerobiosis↗
PubMed1983

Purification and comparative properties of NADPH-cytochrome P-450 reductase from rat and rainbow trout: differences in temperature optima between reconstituted and microsomal trout enzymes.

NADPH-cytochrome P-450 reductase has been purified to apparent homogeneity from liver microsomes of beta-naphthoflavone-treated rats and rainbow trout. The apparent monomeric molecular weights were 75,000 and 77,000 for the rat and trout, respectively. Differences in amino acid composition were observed, particularly for lysine, glycine, threonine, and tyrosine. Analysis of the flavin composition showed that there were 0.97 mol of FAD and 0.92 mol of FMN per mol of rat reductase, whereas the values for the trout enzyme were 1.06 and 0.76 for FAD and FMN, respectively. Trout NADPH-cytochrome c reductase was inhibited by anti-rat antibody, but not to the same extent as was the rat enzyme. No precipitin lines between the trout reductase and rat antibody were observed on Ouchterlony plates. Peptide patterns, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, following limited proteolysis were also markedly different. The trout enzyme was as effective, catalytically, as the rat enzyme in a reconstituted system that contained purified rat cytochrome P-448 and lipid. Comparison of ethoxyresorufin-O-deethylase temperature profiles with various combinations of purified trout and rat P-448, reductase, and lipid, in membranous and nonmembranous reconstitution systems, demonstrated that the lower temperature optimum in trout microsomes could only be reproduced when all three trout components were incorporated into liposomes. These results suggest that it is the structural organization of the mixed-function oxidase enzymes and lipid within trout microsomes which were responsible for the lower temperature optimum compared to rat.

Amino Acids↗
PubMedDate not supplied

Mechanical vibration in neonatal transport: a randomized study of different mattresses.

OBJECTIVE: To test the hypothesis that a gel mattress is most effective in attenuating mechanical vibration in neonatal transport, we performed a randomized block study of four mattress combinations (none, foam, gel, gel on foam) using mannequins and an ambulance traveling on fixed routes (city, highway). STUDY DESIGN: Mechanical vibration was assessed by measuring vertical accelerations at two locations: the forehead of a 2000-gm mannequin and the transport incubator base. From time histories of these accelerations, root mean square (RMS) values and power spectral density functions were calculated. The effect of the mattress on the transmission of vibration was determined from ratios of the RMS values at the two locations. An RMS ratio of < 1.0 indicates attenuation, whereas a ratio of > 1.0 indicates accentuation of vibration. From the power spectral density functions, the natural frequency of the system was determined for each mattress combination in relation to the natural frequencies of the ambulance. To determine the effect of the weight of the mannequin on vibration, additional measurements were performed using a 300-gm mannequin. RESULTS: All the observed RMS ratios were > 1. The highest ratios were observed on the city route in the absence of the gel mattress. The gel mattress, used alone or with the foam mattress, in contrast to foam or no mattress, shifted the natural frequency of the system away from the natural frequencies of the ambulance, avoiding a large amplification of vibration. A decrease in the weight of the mannequin caused the gel mattress to be less effective in attenuating vibration. CONCLUSION: A gel mattress, used alone or with a foam mattress, results in the least accentuation of vibration, but vibration in ambulance transport is not attenuated by any of the mattress combinations. The hazard of vibration may be particularly relevant when transporting extremely low birth weight neonates. These findings indicate a need for study and design of more effective devices that can reduce the vibratory stress.

Acceleration↗