Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of omega- and (omega-1)-hydroxylated metabolites of elaidic and oleic acids in human and rat liver microsomes

A peptide that inhibits the human cholesteryl ester transfer protein (CETP) was isolated from hog plasma by ultracentrifugation, two sequential column chromatographies and electroelution from gels. Molecular weight of the peptide was determined to be approximately 3 kDa on the SDS-PAGE. The peptide contained 28 amino acids with an identical sequence to the amino terminus of hog apolipoprotein-CIII except two amino acid residues: -Pro-Glu- at the fifth and sixth amino acids from the amino terminus in the isolated peptide, in contrast to -Leu-Leu- in hog apo-CIII. A peptide synthesized chemically according to the amino acid sequence of the peptide (designated P28) showed approximately the same degree of CETP inhibitory activity as the isolated peptide. Synthetic peptides with different number of amino acids were also tested for CETP inhibition. Among the peptides, the one with 20 amino acid residues (P20) from the amino terminus showed the highest inhibitory activity against the CETP. The peptide appeared to be associated with the hog high-density lipoproteins (HDL), as determined by immunoblot analysis using antibody against P28. The CETP-inhibitory activity of the peptide was examined in vivo using diet-induced hypercholesterolemic rabbits. When the peptide was injected into the rabbits (7-9 mg/kg body weight), approximately 75% CETP activity disappeared from the plasma in 1 h after the injection and the effect lasted up to 30 h. The inhibition of CETP in vivo led to a concomitant decrease in total plasma cholesterol level up to 30% and an increase in the level of HDL-cholesterol up to 32%. The cholesterol concentrations in the rabbit plasma gradually recovered to the initial level after 48 h.     

Analysis of antibiotics by liquid chromatography-mass spectrometry

We examined the time-resolved and steady-state fluorescence quenching of N-acetyl-L-tryptophanamide (NATA) by acrylamide and iodide, over a range of viscosities in propylene glycol. The quenching of NATA by acrylamide and iodide results in heterogeneity of the intensity decay which increases with the quencher concentration. We attribute the complex decays of NATA to transient effects in diffusion and the nature of the fluorophore-quencher interaction. These data were compared using the phenomenological radiation boundary condition (RBC) and distance-dependent quenching (DDQ) models for collisional quenching. We used global analysis of the time-resolved frequency-domain and steady-state data to select between the models. Consideration of both the frequency-domain and steady state data demonstrate that the quenching rate depends exponentially on the fluorophore-quencher distance, indicating the validity of the DDQ model. The rate constants for acrylamide and iodide quenching, at the constant distance of 5 A, were found to be near 10(13) s-1 and 10(9) s-1, respectively. These rates reflect electron transfer and exchange interactions as the probable quenching mechanisms, respectively.     

Characterisation of TNF-alpha-related peptides by high-performance liquid chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry

Ion-spray triple quadrupole mass spectrometry and high-performance liquid chromatography were used to investigate the products from the solid phase synthesis of (H)-Leu-Thr-Glu-Asn-(OH), a TNF-alpha active-site probe. The target sequence was assembled using tert.-butoxycarbonyl (Boc) chemistry in stepwise fashion from the C-terminal on an Boc-Asn-OCH2-Pam-copoly(styrene-divinylbenzene) resin [Pam = 4-(carboxamidomethyl)benzyl ester]. The crude product was deprotected and cleaved from the resin by HF-p-cresol treatment for 1 h at 0 degrees C. HPLC analysis at 214 nm indicated two late-eluting major products and an early-eluting product. Preparative HPLC demonstrated that the early-eluting product contained ca. 80% of the expected recovered sample mass. Each component was then directly analysed by mass spectrometry and tandem mass spectrometry. The early eluting peak was confirmed as the desired LTEN sequence. Synthesis of the same sequence using 9-fluorenyl methoxycarbonyl (Fmoc) chemistry gave an identical product and confirmed the above analysis. The most significant by-product was derived from arylation of the glutamyl group by the quencher p-cresol. The likely origins of the by-products are discussed.     

Ion-pair high-performance liquid chromatographic separation of two thyroxine glucuronides formed by rat liver microsomes

A simple reversed-phase ion-pair high-performance liquid chromatographic separation method has been developed for thyroxine (T4) and its glucuronide metabolites formed by liver microsomes of untreated and 3-methylcholanthrene-treated rats. Besides the phenol-T4-glucuronide, another, probably acyl-T4-glucuronide, formation has been detected. The effect of pH and temperature on the stability of the acyl-T4-glucuronide was also investigated. The lowering of pH to 2 and cooling the samples to 5 degrees C is necessary to prevent the hydrolysis of acylglucuronide, while both pH and temperature do not affect the stability of the phenol-T4-glucuronide. The retention times of T4 and phenol-T4-glucuronide are highly influenced by the pH of the mobile phase, but not that of acyl-T4-glucuronide.     

Regio- and stereoselective propranolol metabolism by 15 forms of purified cytochromes P-450 from rat liver

Regio- and stereoselectivity of cytochrome P-450-mediated propranolol metabolism (4-, 5- and 7-hydroxylations and N-desisopropylation) was studied using 15 purified cytochrome P-450 species. With each purified cytochrome P-450 species, the regioselectivity was distinct and different between the two optical isomers used as substrates. The stereoselectivity was different depending on the position of propranolol to be metabolized. The regio- and stereoselectivity was altered when substrate concentration was altered, suggesting that the kinetics of the reactions are different depending on the positions of propranolol to be metabolized. Furthermore, the selectivity and its manner of alterations with substrate concentrations were different among all cytochrome P-450 species used. Propranolol, with its multiple metabolic pathways and optical isomers, is an extremely interesting substrate for characterization of cytochrome P-450 species.     

Involvement of CYP2D1 in the metabolism of carteolol by male rat liver microsomes

1. The metabolism of carteolol, a beta-adrenoceptor blocking drug, was investigated in male Sprague-Dawley rat liver microsomes. 2. The formation of 8-hydroxycarteolol was the principal metabolic pathway of carteolol in vitro and followed Michaelis-Menten kinetics with a K(m) = 11.0 +/- 5.4 microM and a Vmax = 1.58 +/- 0.64 nmol/min/nmol P450 respectively (mean +/- SD, n = 5). Eadie-Hofstee plot analysis of carteolol 8-hydroxylase activity confirmed single-enzyme Michaelis-Menten kinetics. 3. The cytochrome P450 isoforms involved in 8-hydroxylation of carteolol were investigated using selective chemical inhibitors and polyclonal anti-P450 antibodies. Quinine (Ki = 0.06 microM) and quinidine (Ki = 2.0 microM), selective inhibitors of CYP2D1, competitively inhibited 8-hydroxycarteolol formation. Furthermore, only anti-human CYP2D6 antibody inhibited this reaction. 4. These results suggest that carteolol is metabolized to 8-hydroxycarteolol by CYP2D1. The K(m) of carteolol for CYP2D1 in male rat liver microsomes was much greater than those of propranolol or bunitrolol, indicating that carteolol has a lower affinity for CYP2D1 compared with these other beta-adrenoceptor blocking drugs.     

Metabolism of nicotine by human liver microsomes: stereoselective formation of trans-nicotine N'-oxide

Liver microsomes from humans catalyze the NADPH-dependent oxidation of (S)-nicotine. The principal product is the 5'-carbon atom oxidation product, nicotine delta 1',5'-iminium ion, which is efficiently converted to the gamma-lactam derivative cotinine in the presence of aldehyde oxidase. Another major product is nicotine N'-oxide. In contrast to previous reports describing in vitro or in vivo studies, formation of only trans-nicotine N'-oxide was observed. Demethylation of nicotine was not observed. Studies on the biochemical mechanism of nicotine 5-carbon atom oxidation strongly implicate one major cytochrome P-450 isoenzyme (i.e., P-450 2A6) as largely responsible for delta 1',5'-iminium ion formation. Stereoselective formation of trans-nicotine N'-oxide may be catalyzed in large part by the flavin-containing monooxygenase (form II). These conclusions are based on the effects of alternate substrates for the flavin-containing monooxygenase, heat inactivation studies, immunoblot studies, and selective substrates for cytochromes P-450. The results suggest that (S)-nicotine trans N'-oxygenation and delta 1',5'-iminium ion formation may be selective probes of human liver flavin-containing monooxygenase form II and cytochrome P-450 2A6 activities, respectively, useful for in vivo phenotyping of humans.     

Future potential of targeted component analysis by multidimensional liquid chromatography-mass spectrometry

Multidimensional liquid chromatography (MDLC) may be used in either (i) the profiling mode where it is the objective to fractionate all components in a mixture or (ii) the targeted component mode in which it is the objective to determine specific analytes. This paper focuses on targeted component analysis from complex mixtures, addressing the critical operations of analyte selection and transport from the first to the second dimension. Although the physical operation of switching a component into the second dimension with computer controlled valving is simple, it is shown that changes in analyte retention time and peak width with column age and fouling are a serious problem. The analyte moves out of the preselected time window for valve switching and quantitation is compromised in the second dimension. It is proposed that a solution to the "drifting peak" phenomenon in targeted component analysis is to use binary mobility elution in the first dimension. Binary mobility refers to those systems, such as affinity chromatography, in which analyte mobility is generally either 0 or 1 relative to mobile phase velocity. Coupling these binary changes in analyte mobility in the first dimension with valve switching eliminates the "drifting peak" phenomenon. In addition, it is shown that a wide time window may be used in affinity separations without compromising the separation or accumulating contaminants. Several cases are described in which immunosorbents were used with reversed phase columns to provide quantitative targeted component analyses from complex mixtures.     

Determination of sedatives and anesthetics in plasma by liquid chromatography-mass spectrometry with a desalting system

Though liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for analysis of drugs and their metabolites, it does not allow the use of a non-volatile buffer such as phosphate buffer. We used a column-switching desalting system in combination with atmospheric pressure chemical ionization LC-MS for analysis of sedatives and anaesthetics. The drugs examined were flumazenil, butorphanol, midazolam, lorazepam, phenobarbital and flunitrazepam. The separation was carried out on a reversed-phase column using acetonitrile-0.1 M phosphate buffer as a mobile phase. The mass spectra are almost the same as those obtained by direct analysis and the molecular ions were clearly observed. In the analysis, phosphate buffer was completely removed with the trapping column and did not interfere with ionization of the drugs in MS. The chiral separation of lorazepam was achieved on a chiral column with UV, optical rotatory detection and MS. This method is sufficiently sensitive and accurate for the pharmacokinetic studies of these drugs in biological samples.     

Direct analysis of several Fusarium mycotoxins in cereals by capillary gas chromatography-mass spectrometry

This article describes three experiments relating to the legibility of TV menus. Special emphasis is placed on the influence of a relatively new feature in TVs: the possibility to blend graphics and video. Three experiments are presented: one concentrating on the influences of blending level and video content; one concentrating on the influences of content and of colour combinations; and one concentrating on the influences of various font characteristics. The results are interpreted in terms of guidelines for blended TV menus.     

In vitro metabolism of clenbuterol and bromobuterol by pig liver microsomes

1. Clenbuterol (CBL) and bromobuterol (BBL) were both extensively metabolized by hepatic microsomes of swine to only one polar metabolite which was separated by hplc and purified to perform mass analysis. 2. LC-MIS analysis by direct infusion into an ion trap system and after reverse-phase chromatograpy into a triple quadrupole system showed that the metabolites were the hydroxylamine-derivatives of CBL and BBL. GC-MS analysis by the CI and EI modes confirmed that the hydroxyl group was bound to the aniline nitrogen. The chemical instability of those metabolites probably as a consequence of spontaneous oxidation and reduction gave rise during the analysis to the corresponding nitroso and nitro derivatives, together with the original compound. 3. Thermal inactivation and CO complex formation were used selectively to inactivate flavin monooxygenase and cytochrome P450, respectively. Both inactivation procedures significantly reduced the formation of the hydroxyl metabolite.     

Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography-mass spectrometry

Laser Doppler flowmetry (LDF) is a sensitive method for the measurement of microvascular blood flow in tissue. The method has been found useful for estimating skin, liver, or gastrointestinal blood flow. Whether it can be applied laparoscopically and whether it is able to measure the intraparenchymal blood flow of an intraabdominal organ is still unknown. In a pilot study, 6 pigs received a laparotomy for placement of a 19-gauge LDF needle probe into the renal parenchyma. Three different locations of the lower pole kidney were chosen for the blood flow measurement. The reliability of using the instrument to measure the renal tissue blood flow was assessed by comparison of the results of renal arterial blood flow obtained from a well-established methodology--ultrasonic Doppler flowmetry. Recordings were taken following (a) intravenous administration of 0.005 mg/kg norepinephrine, (b) manual compression of the suprarenal aorta, and (c) intravenous injection of a lethal dose of phenobarbital (50 mg/kg). Measurements of LDF were possible in all kidney units. The renal tissue perfusion detected by LDF correlated excellently with the renal arterial blood flow under different renal perfusion pressures. The feasibility of using LDF probe to measure the renal tissue perfusion in a laparoscopic model was then assessed in 15 pigs. Under pneumoperitoneum, the right kidneys were approached transperitoneally with the animal in the decubitus position. A total of three trocars were used. The peritoneum and Gerota's fascia were incised and the LDF needle probe was manipulated and inserted by an endoforceps into the renal tissue via a 5-mm trocar. The insertion of the LDF needle probe was technically feasible in all 15 kidney units, and the depth of insertion could be adjusted under direct vision. Baseline values for the renal cortical and renal medullary blood flow were 50.1 +/- 17.7 and 8.8 +/- 3.3 ml/min/100 g tissue, respectively. Spatial variations of the LDF measurements averaged 6%, and temporal variations over 15 min averaged 5%. Four additional hemodynamic parameters were simultaneously recorded, including left carotid artery blood flow, aortic blood pressure, inferior vena caval pressure, and intraabdominal pressure. It appears that systemic and renal hemodynamic parameters can be monitored reliably and continuously in the porcine model. This method allows further information concerning hemodynamic changes and safety of laparoscopy to be obtained.     

Determination of medetomidine, atipamezole and midazolam in pig plasma by liquid chromatography-mass spectrometry

Medetomidine, atipamezole and midazolam in pig plasma were determined by liquid chromatography-mass spectrometry (LC-MS) with an atmospheric pressure chemical ionization interface system by the use of detomidine as an internal standard. The method was applied to studies of pharmacokinetic behaviour of these drugs.     

Enantiomeric separation of denopamine by capillary electrophoresis and high-performance liquid chromatography using cyclodextrins

Direct separation of enantiomers of denopamine was investigated by two separation methods. One is capillary zone electrophoresis (CZE) using cyclodextrins (CDs) (CD-CZE) and the other is high-performance liquid chromatography (HPLC) using CD immobilized chiral stationary phases (CD-CSPs). Enantiomers of denopamine were successfully resolved by employing heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) and acetyl-beta-cyclodextrin (AC-beta-CD), and partially resolved with heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TM-beta-CD), hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and beta-CD polymer under acidic conditions. Separation of enantiomers of denopamine by HPLC was also successful with one of the CD-CSPs, where perphenylated beta-CD (Ph beta-CD) was immobilized. In CD-CZE, some polymeric additives, such as hydroxy-propylmethylcellulose (HPMC) and polyvinylalcohol (PVA), and a coated capillary were used to improve the enantioseparation of denopamine. Method validations such as linearity, recovery and repeatability, etc., were investigated for HPLC, and the method was applied to the optical purity testing of the drug substances and in tablet form.     

Glucuronidation of N-hydroxy heterocyclic amines by human and rat liver microsomes

The food-borne carcinogenic and mutagenic heterocyclic aromatic amines undergo bioactivation to the corresponding N-hydroxy (OH)-arylamines and the subsequent N-glucuronidation of these metabolites is regarded as an important detoxification reaction. In this study, the rates of glucuronidation for the N-OH derivatives of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) by liver microsomal glucuronosyltransferase were compared to that of the proximate human urinary bladder carcinogen, N-OH-aminobiphenyl (N-OH-ABP) and the proximate rat colon carcinogen N-OH-3,2'-dimethyl-4-amino-biphenyl (N-OH-DMABP). Human liver microsomes catalyzed the uridine 5'-diphosphoglucuronic acid (UDPGA)-dependent glucuroidation of N-OH-IQ, N-OH-PhIP, N-OH-Glu-P-1 and N-OH-MeIQx at rates of 59%, 42%, 35% and 27%, respectively, of that measured for N-OH-ABP (11.5 nmol/min/mg). Rat liver microsomes also catalyzed the UDPGA-dependent glucuronidation of N-OH-PhIP, N-OH-Glu-P-1 and N-OH-IQ at rates of 30%, 20% and 10%, respectively of that measured for N-OH-DMABP (11.2 nmol/min/mg); activity towards N-OH-MeIQx was not detected. Two glucuronide(s) of N-OH-PhIP, designated I and II, were separated by HPLC. Conjugate II was found to be chromatographically and spectrally identical with a previously reported major biliary metabolite of PhIP in the rat, while conjugate I was identical with a major urinary metabolite of PhIP in the dog. Hepatic microsomes from rat, dog and human were found to catalyze the formation of both conjugates. The rat preferentially formed conjugate II (I to II ratio of 1:15), while the dog and human formed higher relative amounts of conjugate I (I to II ratio of 2.5:1.0 and 1.3:1.0 respectively). Fast atom bombardment mass spectrometry of conjugates I and II gave the corresponding molecular ions and showed nearly identical primary spectra. However, collision-induced spectra were distinct and were consistent with the identity of conjugates I and II as structural isomers. Moreover, the UV spectrum of conjugate I exhibited a lambda max at 317 nm and was essentially identical to that of N-OH-PhIP, while conjugate II was markedly different with a lambda max of 331 nm. Both conjugates were stable in 0.1 N HCl and were resistant to hydrolysis by rat, dog and human liver microsomal beta-glucuronidases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stereochemical aspects of 1,3-butadiene metabolism and toxicity in rat and mouse liver microsomes and freshly isolated rat hepatocytes

In the last decade, silver staining of nucleolar organizer region-associated proteins (AgNORs) has been widely used in tumour pathology both for diagnostic and for prognostic purposes. However, a reliable and reproducible assessment of these proteins on routinely processed archival tissues has only become possible since the recent introduction of standardized staining method and computer-aided morphometric analysis. In the present study, the AgNOR content at the invasive front of 80 squamous cell carcinomas of the floor of the mouth/tongue was investigated using this novel approach, with regard to prognosis and a variety of clinico-pathological parameters. All standardized AgNOR parameters [mean of AgNOR number, mean of AgNOR area, coefficients of variation (CV) of both AgNOR number and area] were statistically significantly associated with the clinical course. The strongest correlation was found for the AgNOR-area univariate analysis (P = 0.006). In multivariate analysis, the mean of AgNOR number could independently predict both overall (P = 0.01) and disease-free survival (P = 0.001). It is concluded that standardized staining and computer-aided analysis of AgNORs are prerequisites for an objective and reproducible AgNOR assessment, which has potential as a supplementary diagnostic and prognostic tool in oral cancer.     

Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry

A protein with a molecular mass of approximately 62.10(3), derived from open reading frame 2 (ORF-2) of the hepatitis E virus (HEV: Burma strain), was expressed in a baculovirus expression vector and purified to homogeneity. The recombinant 62 kDa protein appeared to be a doublet, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tryptic digestion in conjunction with laser desorption mass spectrometry (LD-MS) and sequence analysis of the tryptic peptides indicated that the amino terminus was blocked, although no proteolytic degradation occurred. The determined internal sequences of peptides were in agreement with the predicted ORF-2 protein. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS) resolved the doublet proteins into two major components with molecular masses of 56548.5 and 58161.4. Confirmation of the amino terminus of the molecule by LD-MS post-ion decay enabled us to tentatively assign the carboxyl terminus of each species at residues 540 and 525. Sequencing of the intact protein by automated carboxyl terminal sequencing confirmed that the carboxyl terminus was truncated and that the sequence assignment predicted by LC-MS was correct.     

P-450-dependent metabolism of lauric acid in alcoholic liver disease: comparison between rat liver and kidney microsomes

Cytotoxic T lymphocytes (CTL) are potent effector cells that could provide long term antitumor immunity if induced by appropriate vaccines. CTL recognize 8-14 amino acid-long peptides processed intracellularly and presented by MHC class I molecules. A well-characterized example of a potential tumor antigen in childhood pre-B Acute Lymphoblastic Leukemia (ALL) results from the chromosomal translocation 12;21 leading to the fusion of the ETV6 and AML1 genes. This translocation is observed in > 25% of ALL-patients. In this study, we have examined whether the chimeric ETV6-AML1 protein could serve as a tumor specific antigen for CTL in HLA-A2.1 individuals. We have identified a nonapeptide (RIAECILGM), encoded by the fusion region of the ETV6-AML1 protein, that binds to HLA-A2.1 molecules and induces specific primary CTL in peripheral blood lymphocytes from healthy donors. These CTL specifically lysed HLA-A2.1 tumor cells endogeneously expressing the ETV6-AML fusion protein. CTL with similar functional capacities were found with high frequencies and cloned from one patient's bone marrow indicating that ETV6-AML1-specific anti-ALL CTL are, at least in some patients, spontaneously stimulated and might participate to host antileukemia defense.     

Separation of tetracyclines by high-performance capillary electrophoresis and capillary electrochromatography

Separations of various tetracycline mixtures by high-performance capillary electrophoresis (HPCE) and a new form of electrochromatography (CEC) are compared. The new CEC method involves etching the inner wall of the capillary surface with an appropriate reagent (ammonium dihydrogen fluoride) in order to produce a significant increase in surface area. The etched surface is then modified by a silation/hydrosilation reaction sequence to first produce a hydride intermediate which is then further reacted to attach a C18 moiety. The bare and hydride capillaries are tested under HPCE conditions while the C18 capillary functions in the CEC mode. The effects of pH and the presence of an organic modifier (methanol) are also studied. Detection limits ( < 10 micrograms/ml) are comparable to previous HPLC and HPCE results. Resolutions for mixtures which simulate real analytical problems are equal to or better than those reported for separations on polymeric and diol columns by HPLC and in earlier studies by HPCE and MECC.