Selectivity in zeolite occlusion of olefines.
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Alkalies↗
Biomedical subjects
C Fenselau
Publications and source records attributed to C Fenselau.
Characterization of intact microorganisms by MALDI mass spectrometry.
The application of MALDI mass spectrometry to desorb protein biomarkers from intact viruses, bacteria, fungus, and spores is the focus of this review. Instrumentation, sample collection, sample preparation, and algorithms for data analysis are summarized. Optimally these analyses should be carried out in less than five minutes. Successful applications are discussed from biotechnology, cell biology, and the pharmaceutical industry.
Biomarkers↗
Studies of intramolecular rearrangements of acyl-linked glucuronides using salicylic acid, flufenamic acid, and (S)- and (R)-benoxaprofen and confirmation of isomerization in acyl-linked delta 9-11-carboxytetrahydrocannabinol glucuronide.
NMR and HPLC have been used to investigate the rearrangements of 1-O-acylglucuronides in vitro and the occurrence of rearranged isomers in urine. Glucuronides of flufenamic acid, (S)- and (R)-benoxaprofen, salicylic acid, and delta 9-11-carboxytetrahydrocannabinol were synthesized, by use of immobilized enzymes, or purified from urine. Ester-linked isomers of these gluruconides were characterized, and isomers derived from flufenamic acid, (S)-benoxaprofen, and salicylic acid were purified for further study by NMR and HPLC. The positions of the new ester linkages could be identified by two-dimensional NMR. Shifts not only in the resonance of the proton adjacent to the esterified hydroxyl group but also in the resonance of the anomeric proton on carbon 1 of the glucuronic acid moiety could be correlated with the position of each isomeric ester bond. HPLC elution times also correlated with ester position in this small set of samples. The sequences of isomer formation were studied in situ by NMR and also at pH 8 by HPLC. These studies indicate that, for the three cases examined, the C-2 ester is formed first, followed by formation of C-3 and C-4 esters. The purified isomeric esters were found not to re-form the high-energy 1-O-acyl bond. All other rearrangement steps are reversible. In contrast to other glycosides and glycerol esters, no evidence could be found for rearrangements beyond nearest-neighbor hydroxyl groups in glucuronic acid. The sequence of formation and reversibility is consistent with an ortho ester intermediate, as has been proposed for rearrangements of other glycosides.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
High-performance tandem mass spectrometry in metabolism studies.
1. High-performance tandem mass spectrometry provides unit resolution in both selection of precursor ions and analysis of fragment ions, and extensive and reproducible fragmentation through collisional activation at high energy. 2. Metabolites can be analysed that occur as minor components in h.p.l.c. peaks or other mixtures. Homogeneous isotopic species can be selected for unambiguous analysis of distributions of isotope labels. Fragmentation may be significantly enhanced to provide structural information. Overall, the signal to noise ratio is greatly improved and the spectrum is simplified. 3. These points are illustrated by isotope-labelling studies of the mechanisms of glutathione conjugation of the anti-tumour agent cyclophosphamide, the cytotoxic agent phosphoramide mustard and dimethylbilirubin, an analogue of bilirubin designed to be distinguishable from endogenous bilirubin. Analysis of isomeric mixed disulphides formed between glutathione and a peptide with an internal disulphide bond is discussed. 4. Reaction-induced decomposition is presented as an alternative to collisionally induced decomposition with more efficient energy transfer.
Animals↗
Glutathione conjugation with phosphoramide mustard and cyclophosphamide. A mechanistic study using tandem mass spectrometry.
The conjugations of cyclophosphamide and of phosphoramide mustard with glutathione are shown to be catalyzed by hepatic cytosolic glutathione-S-transferases. Cyclophosphamide conjugation is also catalyzed by microsomal glutathione-S-transferases, both in intact microsomes and after solubilization and immobilization. Deuterium isotope labels are used to test whether chloride is directly displaced by glutathione in the enzyme-catalyzed conjugations, or whether conjugation takes place via symmetrical cyclic aziridinium ions. Tandem mass spectrometry with high energy collisional activation is shown to provide reliable analysis of the isotope-labeling patterns in the conjugated products. This experiment leads to the conclusion that the aziridinium ion is opened in the conjugation of phosphoramide mustard in both the enzyme-catalyzed and the chemical reactions. Cyclophosphamide, on the other hand, is shown to be conjugated through direct displacement of chloride.
Animals↗
Acylation of albumin by 1-O-acyl glucuronides.
Representative examples of drug metabolites containing a carboxylic acid group conjugated to glucuronic acid are shown to be active chemical electrophiles, which acylate albumin in vitro through transesterification reactions. Based on these and other observations, we propose that this acylating reactivity is characteristic of 1-O-acyl glucuronides as a class, and that albumin is representative of many susceptible biopolymers. The hypothesis is advanced that this reaction can occur in vivo as well, and that it may offer a molecular mechanism for the physiological activity or toxicity of some xenobiotic and lipophilic compounds.
Acylation↗
Glutathione conjugates. Immobilized enzyme synthesis and characterization by fast atom bombardment mass spectrometry.
Glutathione transferase activity was shown to be present in an immobilized preparation of microsomal protein. Chlorodinitrobenzene, ethacrynic acid, captopril, styrene oxide, and iminocyclophosphamide were found to be substrates, each providing a different kind of electrophilic functional group for conjugation. The glutathione conjugates were characterized by thin layer chromatography (visualized by reaction with ninhydrin) and by high pressure liquid chromatography. A variety of conditions was evaluated for analysis of these glutathiones by fast atom bombardment mass spectrometry.
Animals↗
Conversion of melphalan to 4-(glutathionyl)phenylalanine. A novel mechanism for conjugation by glutathione-S-transferases.
One of the conjugates of melphalan, characterized following incubation with glutathione (GSH) and immobilized microsomal glutathione-S-transferases, has been identified as 4-(glutathionyl)-phenylalanine. This conjugate is formed by displacement of the mustard moiety. The structure was confirmed by reaction of the corresponding 4-halophenylalanines with GSH as well as by TLC, HPLC, and FAB mass spectrometry. Evidence is presented here to support the hypothesis that this novel reaction occurs via a cyclic aziridinium ion. To test this proposed mechanism, N,N-dimethyl-p-toluidine and its corresponding quaternary ammonium iodide salt were incubated with GSH in the presence of immobilized glutathione-S-transferases at 37 degrees C for 1 hr at pH 7.4. The tertiary amine did not react, whereas the quaternary compound produced 4-(glutathionyl)toluene. The effect of ring substituent requirements for the reaction was evaluated. The formation of GSH adducts of alkylating agents may be a factor in the development of resistance to these drugs.
Animals↗
Species-dependent glucuronidation of drugs by immobilized rabbit, rhesus monkey, and human UDP-glucuronyltransferases.
The utility of immobilized microsomal enzymes from rabbit liver for the synthesis of a variety of conjugated drug metabolites has been demonstrated in our laboratory. Here, this capability is extended to microsomal preparations from monkey and human liver. A comparative study of glucuronidation of various substrates was carried out using immobilized uridine-5'-diphosphoglucuronyltransferase from rabbit, rhesus monkey, and human liver. The three drugs used for the study (sulfadimethoxine, oxazepam, and cyproheptadine) were chosen on the basis of their previously reported in vivo species differences in glucuronidation. Aglycons were incubated with UDP-glucuronic acid in the presence of immobilized UDP-glucuronyl-transferase at pH 7.4 for 16 hr at 37 degrees C. Glucuronide products were purified by high performance liquid chromatography and further characterized by thin layer chromatography and mass spectometry. Species-dependent glucuronidation was observed in all three cases. Differences in the in vitro product formation paralleled in vivo species differences for the three drugs studied. The same glucuronides were produced by immobilized human liver microsomes as have been found to be formed in vivo from the three drugs studied.
Animals↗
Synthesis and characterization of glucuronides of 5'-hydroxy-delta9-tetrahydrocannabinol and 11-hydroxy-delta9-tetrahydrocannabinol.
Glucuronides of two metabolites of delta9-tetrahydrocannabinol, 11-hydroxy-delta9-THC and 5'-hydroxy-delta9-THC, have been synthesized and characterized. Two isomeric monoglucuronides were isolated from the incubation of each metabolite with UDP-glucuronyltransferase immobilized on beaded Sepharose. The structures of these conjugates were assigned primarily on the basis of combined gas chromatography-mass spectrometry.
Animals↗
Species-dependent enantioselective glucuronidation of three 2-arylpropionic acids. Naproxen, ibuprofen, and benoxaprofen.
The enantioselective glucuronidation of several racemic 2-arylproprionic acids (naproxen, ibuprofen, and benoxaprofen) was investigated in vitro with immobilized microsomal protein from human, rhesus monkey, and rabbit liver as the source of UDP-glucuronyl-transferases. Human microsomes, solubilized microsomal protein, and immobilized protein all gave comparable enantioselectivity. The diastereomeric glucuronides were separated and quantitated by HPLC and characterized stereochemically by co-elution with glucuronides formed from authentic resolved enantiomers. Conjugation of the carboxylic acid moieties occurred stereoselectively with all three substrates. However, enantioselectivity varied qualitatively and quantitatively with substrate as well as with species. The glucuronidation of (S)-naproxen by human liver enzymes was inhibited in the presence of (R)-naproxen and vice versa. The ratio of the glucuronides of (S)-benoxaprofen to that of (R)-benoxaprofen in rhesus monkey urine varied between individual animals and appeared to change through time as dosing continued. Hydrolysis of the diasteriomeric glucuronides of (R)- and (S)-naproxen was differentially inhibited by addition of 1,4-saccharolactone.
Animals↗
Enzymatic conjugation and hydrolysis of [18O]isoborneol glucuronide.
Introduction of 18O into the acetal linkage of isoborneol glucuronide has been accomplished through the in vitro enzymatic conjugation of 18O-labeled isoborneol by use of immobilized, partially purified, rabbit liver UDP-glucuronyltransferase supplemented with UDP-glucuronic acid. Mass-spectrometric analysis of 18O enrichment in both the aglycon and the intact conjugate made it possible to study both enzymatic conjugation and enzymatic hydrolysis. Analysis by gas chromatography-mass spectrometry indicated that the labeled hydroxyl oxygen of isoborneol is retained during both conjugation and hydrolysis.
Animals↗
Analysis of intact glucuronides by mass spectrometry and gas chromatography-mass spectrometry. A review.
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Acetylation↗
Simultaneous immobilization of cytochrome P-450 and glucuronyltransferase for synthesis of drug metabolites.
Cytochrome P-450, NADPH-cytochrome P-450 reductase, and glucuronyltransferase were immobilized simultaneously on cyanogen bromide-activated Sepharose from phenobarbital-induced rabbit liver microsomes. The activity of the P-450 system was demonstrated by the N-demethylation of ethylmorphine and the O-demethylation of p-nitroanisole. p-Nitrophenol produced from the oxidation of p-nitroanisole was conjugated to p-nitrophenyl glucuronide, as evidenced by isolation and characterization of the glucuronide by GC/MS.
Animals↗
Stable isotope analysis by fast-atom bombardment labeling of UDP-glucuronic acid.
Fast-atom bombardment mass spectrometry is found to provide a method for analysis of isotopes in the enzyme cofactor uridine-5'-diphosphoglucuronic acid, heretofore unsusceptible to mass-spectral characterization. This technique was used to determine optimal conditions for the introduction of 18O by acid-catalyzed exchange in H218O and to evaluate the loss of the isotope when labeled cofactor is used in enzymatic incubations. Fast-atom bombardment mass spectrometry provided a quantitative assessment of various isotopic species and also permitted the location of the isotopes in the molecule to be determined.
Mass Spectrometry↗
Synthesis of quaternary ammonium-linked glucuronides by rabbit hepatic microsomal UDP-glucuronyltransferase and analysis by fast-atom bombardment mass spectrometry.
The quaternary ammonium-linked glucuronides of tripelennamine and cyproheptadine, important human metabolites of these drugs, were synthesized with an immobilized rabbit hepatic microsomal enzyme system as catalyst. The glucuronide of pyridine was prepared by chemical synthesis. These cationic, thermally labile, quaternary ammonium-linked glucuronides were analyzed by fast-atom bombardment mass spectrometry. The mass spectra contained intense molecular cations and characteristic losses of neutral fragments. The information contained in the spectra was sufficient to characterize the difficult-to-analyze intact quaternary ammonium glucuronides and provide structural information about the aglycon.
Animals↗
Metabolic formation of N- and O-glucuronides of 3-(p-chlorophenyl)thiazolo[3,2-a]benzimidazole-2-acetic acid. Rearrangement of the 1-o-acyl glucuronide.
Excretion of 3-(p-chlorophenyl)thiazolo[3,2-a]benzimidazole-2-acetic acid (I) and its metabolites was studied in rats, beagle dogs, and rhesus monkeys given 20-mg/kg doses of 14C-labeled drug. The urine of rhesus monkeys contained two metabolites in addition to unchanged drug. Both metabolites were hydrolyzed to I by beta-glucuronidase and the hydrolysis was inhibited by 1,4-saccharolactone, indicating that they were glucuronides of I. One of the metabolites (III) was not hydrolyzed by dilute alkali. Its NMR spectrum indicated that the site of conjugation was one of the nitrogen atoms, i.e., it was a quaternary N-glucuronide. The FAB mass spectrum was in conformity with this assignment. This metabolite was not present in the urine of dogs or rats given labeled drug. The other metabolite (II) was excreted in the urine of all three species as well as in the bile of the rat. It was readily hydrolyzed by dilute alkali (pH 11 for 0.5 hr at 37 degrees C), indicating that this metabolite was an acyl glucuronide. The metabolite was stable at pH 4.5 but it was readily converted to three isomers at 37 degrees C within 1 hr at pH 6.5 and above. The mass spectra of the derivatized isomers and metabolite were similar. The isomers were hydrolyzed to I by dilute alkali but not by beta-glucuronidase. They exhibited reducing properties (whereas metabolite II did not), suggesting that they were formed by acyl migration of the aglycone to the second, third, and fourth carbon atoms of the glucuronic acid moiety. Acyl migration probably plays a role in the disposition of I as well as other drugs that form labile glucuronides.
Animals↗
Electrophilic reactions of acyl-linked glucuronides. Formation of clofibrate mercapturate in humans.
An acyl-linked mercapturic acid metabolite of clofibrate has been identified in human urine. The identification is based on comparison of fast-atom bombardment mass spectra and high-pressure liquid chromatograms with those of synthesized material. This represents the first acyl-linked mercapturate found in man. The mechanism proposed for its formation involves transacylation of clofibrate acyl glucuronide by glutathione, hydrolysis, and acetylation to produce clofibryl mercapturic acid. Synthetic clofibrate glucuronide is shown to be electrophilic; it reacts covalently with ethanethiol and p-nitro-benzylpyridine. We propose that clofibrate acyl glucuronide is an electrophilic metabolite which reacts with sulfhydryl groups and therefore may be responsible for the human hepatotoxicity of clofibrate.
Acylation↗