An ion exchange liquid chromatography/mass spectrometry method for the determination of reduced and oxidized glutathione and glutathione conjugates in hepatocytes.

A rugged LC-MS/MS method was developed to quantify reduced and oxidized glutathione (GSH and GSSG, respectively) in rat hepatocytes. In addition, GSH conjugates can be detected, characterized and measured in the same analysis. Samples were treated with acetonitrile and iodoacetic acid to precipitate proteins and trap free GSH, respectively. These highly polar analytes were separated by ion exchange chromatography using conditions that were developed to be amenable to electrospray ionization and provide baseline chromatographic resolution. A solvent gradient with a total run time of 13 min was used to elute the analytes, as well as any highly retained components in the samples that would otherwise accumulate on the HPLC column and degrade the chromatography. The analytes were detected using either selected ion monitoring (SIM) using an ion trap mass spectrometer or selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer. The ranges for quantification of GSH and GSSG using an ion trap were 0.651-488 microM and 0.817-327 microM, respectively. Using SRM with the triple quadrupole instrument, the ranges of quantification for GSH and GSSG were 0.163-163 microM and 0.0816-81.6 microM, respectively. The accuracy and precision for both methods were within 15%. The utility of the method was demonstrated by treating rat hepatocytes with model compounds menadione and precocene I. Menadione, which contains a quinone moiety that undergoes redox cycling and induces concentration- and time-dependent oxidative stress in hepatocytes, resulted in decreased GSH concentrations with concomitant increase in concentrations of GSSG, as well as a GSH-menadione conjugate. When hepatocytes were incubated with precocene I, a time-dependent decrease in GSH concentrations was observed with concomitant increase in a GSH-precocene conjugate. GSSG concentrations did not increase in the presence of precocene I, consistent with its lack of redox activity. This analytical method has general utility for simultaneously investigating the potential of test compounds to induce both oxidative stress from redox cycling in vitro and the formation of GSH conjugates.

Metabolism of carvedilol in dogs, rats, and mice.

The excretion and biotransformation of carvedilol [1-[carbazolyl-(4)-oxy]-3-[(2-methoxyphenoxyethyl)amino]-2-p ropanol], a new, multiple-action, neurohormonal antagonist that exhibits the combined pharmacological activities of beta-adrenoreceptor antagonism, vasodilation, and antioxidation, were investigated in dogs, rats, and mice. Carvedilol was absorbed well, and biliary secretion was predominant in each species. Carvedilol was metabolized extensively in each species, and elimination of unchanged compound was minor in bile duct-catheterized rats and dogs. In dogs, glucuronidation of the parent compound and hydroxylation of the carbazolyl ring, with subsequent glucuronidation, were the major metabolic pathways. Rats showed the simplest metabolite profile; the primary metabolites were formed by hydroxylation of the carbazolyl ring, with subsequent glucuronidation. Mice displayed the most complicated metabolite profile; glucuronidation of the parent compound and hydroxylation of either the carbazolyl or phenyl ring, with subsequent glucuronidation, were the major metabolic routes. O-Dealkylation was a minor pathway in all species examined.

Application of liquid chromatography-mass spectrometry(n) analyses to the characterization of novel glyburide metabolites formed in vitro.

The application of bench-top ion-trap atmospheric pressure ionization mass spectrometry in the characterization of in vitro metabolites of glyburide is discussed. The metabolites formed in vitro by rat, dog, monkey and human liver microsomes were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by mass spectrometry (MS)n experiments. The utility of data dependent MS1-MS2-MS3 analyses, where the mass spectrometer makes "real-time" decisions about the experiment to be performed, are described using the characterization of two novel metabolites of glyburide as an example. The metabolite profiles from each species were similar. Six cyclohexyl hydroxylation products were detected, as well as two novel monooxygenation products formed via hydroxylation of the ethyl chain at the benzylic position, and alpha to the amide nitrogen. The ion-trap with electrospray ionization proved to be a sensitive and reliable HPLC detection system that provided important chemical structure information.

Direct plasma liquid chromatographic-tandem mass spectrometric analysis of granisetron and its 7-hydroxy metabolite utilizing internal surface reversed-phase guard columns and automated column switching devices.

An alternative on-line automated sample enrichment technique useful for the direct determination of various drugs and their metabolites in plasma is described for rapid development of highly sensitive and selective liquid chromatographic methods using mass spectrometric detection. The method involves direct injection of plasma onto an internal surface reversed-phase (ISRP) guard column, washing the proteins from the column to waste with aqueous acetonitrile, and backflushing the analytes onto a reversed-phase octyl silica column using switching valves. The analytes were detected using a tandem mass spectrometer operated in selected reaction monitoring (SRM) mode using atmospheric pressure chemical ionization (APCI). Use of two ISRP guard columns in parallel configuration allowed alternate injections of plasma samples on these columns for sample enrichment and shortened the column equilibration and LC-MS-MS analysis times, thereby increasing the sample throughput. The total run time, including both sample enrichment and chromatography, was about 6 min. Using this technique, an analytical method was developed for the quantitation of granisetron and its active 7-hydroxy metabolite in dog plasma. Granisetron is a selective 5-HT3 receptor antagonist used in the prevention and treatment of cytostatic induced nausea and vomiting. Recovery of the analytes was quantitative and the method displayed excellent linearity over the concentration ranges tested. Results from a three day validation study for both compounds demonstrated excellent precision (1.3-8.7%) and accuracy (93-105%) across the calibration range of 0.1 to 50 ng/ml using an 80 microliters plasma sample. The automated method described here was simple, reliable and economical. This on-line approach using ISRP columns and column switching with LC-MS-MS is applicable for the quantification of other pharmaceuticals in pharmacokinetic studies in animals and humans which require high sensitivity.

Renal catabolism of recombinant human soluble CD4 after intravenous administration to male Sprague-Dawley rats.

Recombinant soluble CD4 (sT4; mol. wt. 45,000) has been studied extensively in Sprague-Dawley rats, and substantial renal processing has been indicated. In rats and monkeys, renal filtration and precipitation of sT4 in the distal nephron caused tubular cast nephropathy. Intravenous pharmacokinetics in the rat demonstrated that sT4 plasma clearance exceeded the glomerular filtration rate. In an effort to determine quantitatively the extent to which kidney and other tissues were responsible for sT4 catabolism, sT4 was labeled with trace amounts of dilactitol-[125I]tyramine and administered intravenously to Sprague-Dawley rats (1 mg/kg). Dilactitol-tyramine accumulates in lysosomes at the site of protein degradation. It has been used primarily to demonstrate hepatic catabolism of endogenous proteins. Blood samples were drawn for pharmacokinetic analysis, and selected tissues were removed to assess radiolabel distribution. Comparison of pharmacokinetic parameters derived from total plasma radiolabel and functional ELISA were not significantly different. Thus, covalent modification of sT4 with dilactitol-tyramine did not appreciably change the rate of clearance. From 3 to 24 hr after intravenous administration, 81.5 +/- 0.1% of the total administered radioactivity was found in the kidney. Approximately 8-13% of the administered dose was recovered in the liver. Macroscopic autoradiography of the kidney demonstrated accumulation of radiolabel in the cortex. Light microscopic autoradiography of the kidney following intravenous administration of directly radioiodinated sT4 confirmed cortical processing, because radiolabel was located primarily in epithelial cells of P1 and P2 segments of the proximal tubule after low intravenous doses (0.4-4 mg/kg). At 40 mg/kg, distal tubules and cortical collecting ducts were labeled as well. Thus, sT4 was filtered by the glomerulus, reabsorbed in the proximal tubule, and degraded in the lysosomal compartment.

A liquid chromatographic assay for the stereospecific quantitative analysis of halofantrine in human plasma.

A stereospecific liquid chromatographic (LC) assay was developed for the quantification of the antimalarial drug, halofantrine, in human plasma. Following protein precipitation with acetonitrile, the enantiomers of halofantrine were extracted from human plasma using ammonium hydroxide and tert-butyl methyl ether-hexane. A precolumn derivatization step was employed using (+)-di-O-acetyl-L-tartaric acid anhydride to form diastereomeric derivatives of the halofantrine enantiomers. Chromatographic resolution of the diastereomers was performed using reversed-phase LC with UV detection at 254 nm. The recovery of (+/-)-halofantrine from human plasma at 25 and 2000 ng ml-1 was 68.2 and 61.4%, respectively. The derivatization yield following extraction and derivatization of 2000 ng ml-1 of (+/-)-halofantrine was 95.6%. Using 0.5 ml of plasma, the limit of quantification for each halofantrine enantiomer was 12.5 ng ml-1. Linear responses in analyte/internal standard peak height ratios were observed for analyte concentrations ranging from 12.5 to 1000 ng ml-1. Chromatograms of drug-free plasma showed no interfering peaks with retention times similar to those for (+)- and (-)-halofantrine or internal standard. Based on the validation data, the assay performed well over the enantiomer concentration range of 12.5-500 ng ml-1.

Isolation and identification of singlet oxygen oxidation products of beta-carotene.

Singlet oxygen is a highly reactive form of oxygen produced by many toxic photosensitizers. beta-Carotene quenches singlet oxygen catalytically through a very efficient physical reaction. However, concomitant chemical reactions during photosensitized oxidations consume beta-carotene. To investigate the hypothesis that chemical reactions with singlet oxygen consume beta-carotene, we characterized products of the photosensitized oxidation of beta-carotene. beta-Carotene and the photosensitizer rose bengal were dissolved in toluene/methanol (85:15 v/v), which was bubbled with O2 and illuminated with a quartz-halogen lamp for 30 min at 5 degrees C. Reaction products were analyzed by reverse-phase HPLC, UV-vis spectrophotometry, and mass spectrometry. beta-Carotene oxidation products were identified as beta-ionone, beta-apo-14'-carotenal, beta-apo-10'-carotenal, beta-apo-8'-carotenal, and beta-carotene 5,8-endoperoxide. Formation of these products was dependent on the presence of the photosensitizer. The products apparently were formed from the action of singlet oxygen rather than by photochemically-initiated beta-carotene autoxidation, since suppression of autoxidation by equimolar alpha-tocopherol did not diminish product formation. beta-Carotene autoxidation initiated by 2,2'-azobis(2,4-dimethylvaleronitrile), which generates peroxyl radicals, yielded a different product distribution than that from photosensitized oxidation. Specific products formed by singlet oxygen oxidation of beta-carotene may serve as markers for singlet oxygen quenching in biological systems.

Formation of mitochondrial phospholipid adducts by nephrotoxic cysteine conjugate metabolites.

Nephrotoxic cysteine conjugates derived from a variety of halogenated alkenes are enzymatically activated via the beta-lyase pathway to yield reactive sulfur-containing metabolites which bind covalently to cellular macromolecules. Mitochondria contain beta-lyase enzymes and are primary targets for binding and toxicity. Previously, mitochondrial protein and/or DNA have been considered as molecular targets for cysteine conjugate metabolite binding. We now report that metabolites of nephrotoxic cysteine conjugates form covalent adducts with rat kidney mitochondrial phospholipids. Rat kidney mitochondria were incubated with the 35S-labeled conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), S-(1,2-dichlorovinyl)-L-cysteine, and S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine. Quantitation of metabolite binding to whole mitochondria and to mitochondrial protein and lipid fractions revealed that as much as 42% of the 35S-label associated with the mitochondria was found in the lipid fraction. Total lipids were also extracted from 35S-treated mitochondria and separated by thin-layer chromatography. 35S-Containing metabolites were found in the lipid fractions from mitochondria treated with each of the conjugates. Lipids from both [35S]CTFC- and [35S]-TFEC-treated mitochondria contained major 35S-labeled lipid adducts which had similar mobility by thin-layer chromatography. Fatty acid analysis, 19F and 31P NMR spectroscopy, and mass spectrometric analyses confirmed that the major TFEC and CTFC adducts are thioamides of phosphatidylethanolamine.

Purification, primary structure characterization, and cellular distribution of two forms of cellular retinol-binding protein, type II from adult rat small intestine.

Cellular retinol-binding protein type II (CRBP(II)) is a major protein in the small intestine, accounting for more than 1% of the soluble protein recovered from rat jejunal mucosa. Two forms of the protein, called CRBP(II)A and CRBP(II)B, were purified from rat small intestine using a three-column procedure. The two forms were present in equal abundance. The primary structures of CRBP(II)A and CRBP(II)B were determined using a combination of techniques including amino acid composition and sequence analyses, and fast atom bombardment and gas chromatography-electron impact mass spectrometry. The primary structures of both proteins were found to be identical, but they differed in their NH2-terminal processing. CRBP(II)B was acetylated at its NH2 terminus, while CRBP(II)A was not. The results also confirmed the amino acid sequence of CRBP(II)A that was deduced from the cDNA sequence by Li et al. (Li, E., Demmer, L. A., Sweetser, D. A., Ong, D. E., and Gordon, J. I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5770-5783). Antibodies capable of distinguishing between the two forms of CRBP(II) were used for immunohistochemical studies which indicated that the organ and cellular distributions of the two forms were identical. The 50% acetylation observed here in vivo fits the pattern predicted by recent in vitro studies which described the effect of NH2-terminal sequence on cotranslational NH2-terminal processing of cytosolic proteins (Boissel, J. P., Kasper, T. J., and Bunn, H. F. (1988) J. Biol. Chem. 263, 8443-8449). Our results provide a basis for investigating the possibility of different roles of CRBP(II)A and CRBP(II)B within cells, as well as the importance of acetylation of the amino terminus for these biological functions.

Reaction of the model thiol 2-mercaptoethanol and glutathione with methylvinylmaleimide, a Michael acceptor with extended conjugation.

Many alpha,beta-unsaturated compounds are sufficiently reactive to condense with nucleophiles under physiological conditions and are potentially deleterious to cellular processes. These compounds react with thiols by nucleophilic attack to give 1,4 addition products. We have examined the products formed from the reaction of the model thiols HSCH2CH2OH and glutathione with methylvinylmaleimide, a Michael acceptor with extended conjugation. Glutathione produced exclusively a 1,6 addition product with methylvinylmaleimide. HSCH2CH2OH also formed a 1,6 nucleophilic addition product, as well as a disubstituted product resulting from apparent further 1,4 addition to the 1,6 addition product. Two other novel products which resulted from addition to the maleimide ring and addition at the vinyl carbon proximal to the maleimide ring were observed.

A mutant Paramecium with a defective calcium-dependent potassium conductance has an altered calmodulin: a nonlethal selective alteration in calmodulin regulation.

The Paramecium mutant, pantophobiac A, has a defect that results in an in vivo loss of calcium-dependent potassium efflux channel activity. This defect is corrected fully by the microinjection of wild-type Paramecium calmodulin into pantophobiac A cells and is partially restored by calmodulins from other organisms, but it cannot be restored by microinjection of pantophobiac calmodulin. Overall, these results suggested that wild-type Paramecium calmodulin has unique features that allow it to restore fully a normal phenotype and that the defect in pantophobiac A might be an altered calmodulin molecule. Previous studies established the amino acid sequence of wild-type calmodulin and showed that Paramecium calmodulin has several differences from other calmodulins, including the presence of dimethyllysine at residue 13. To test directly the possibility that calmodulin from the pantophobiac mutant might be altered, we purified the mutant calmodulin and compared its properties to those of wild-type Paramecium calmodulin. We found one amino acid sequence difference between the two Paramecium calmodulins: a phenylalanine in the mutant protein, instead of a serine, at residue 101. This change is at a calcium-liganding residue in the third calcium-binding loop. These and previous studies demonstrate that comparatively subtle changes in the structure of calmodulin can result in quantitative alterations in in vivo activity, provide insight into the in vivo roles of calmodulin and the regulation of ion channels, and demonstrate that functional alterations of calmodulin are not necessarily lethal.

Amino acid sequence of a novel calmodulin from Paramecium tetraurelia that contains dimethyllysine in the first domain.

A class of Paramecium behavioral mutants called pantophobiacs have a deficiency in calcium-dependent potassium efflux, and this deficiency can be corrected by the microinjection of wild-type Paramecium calmodulin (Hinrichsen, R. D., Burgess-Cassler, A., Soltvelt, B. C., Hennessey, T., and Kung, C. (1986) Science 232, 503-506). As a starting point in investigations of which features allow wild-type Paramecium calmodulin to fully restore this behavior while other calmodulins are inactive or poorly effective, we elucidated the amino acid sequence of the wild-type calmodulin. We utilized an approach that combined Edman chemistry with mass spectrometry. This approach resulted in the identification of a new post-translational modification in calmodulin: N epsilon,N epsilon-dimethyllysine at residue 13. This particular modification has not been described for calmodulins studied previously. The only other first-domain modification that has been described for any calmodulin is acetylation of the amino terminus (Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) J. Biol. Chem. 255, 962-975). These results along with analyses of pantophobiac calmodulin and calmodulin binding proteins will provide insight into calmodulin's role in a well-defined behavioral mutant.

Characterization of the enzymatic and nonenzymatic peroxidative degradation of iron porphyrins and cytochrome P-450 heme.

Both purified cytochrome P-450 (P-450) and free ferriprotoporphyrin IX are destroyed by NADPH-P-450 reductase in the presence of NADPH and O2. The process appears to be mediated by H2O2 generated by reduction of O2. Six major products were identified from the reaction of H2O2 with ferri-protoporphyrin IX-hematinic acid, methylvinylmaleimide, and four dipyrrolic propentdyopents. The structures of the propentdyopents were elucidated by mass spectrometry and 1H NMR methods. Both free ferriprotoporphyrin IX and P-450 yielded these same products in similar relative ratios. P-450 heme in rat liver microsomes was degraded in the presence of O2 and NADPH and either NaN3 (a catalase inhibitor) or Fe-ADP (which promotes lipid peroxidation); the products were primarily hematinic acid, methylvinylmaleimide, and small quantities of one propentdyopent. Only the two maleimides were detected in the destruction of microsomal P-450 heme by cumene hydroperoxide and iodosylbenzene. On the basis of the reaction of H2O2 with several metal-octaethylethylporphyrin complexes and free octaethylporphyrin, the iron chelated in ferriprotoporphyrin IX is required for degradation by H2O2. Biliverdin is not an intermediate in the formation of maleimides and propentdyopents from heme. Experiments using the tetraethylpropentdyopent produced from ferrioctaethylporphyrin suggest that propentdyopents are not further cleaved to form the maleimides. A mechanism for oxidative heme destruction consistent with these observations is proposed.