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Biomedical subjects

K M Straub

Publications and source records attributed to K M Straub.

At least 19 recordsLinked to original sources
PubMed1995

Identification of a specific methionine in mammalian 15-lipoxygenase which is oxygenated by the enzyme product 13-HPODE: dissociation of sulfoxide formation from self-inactivation.

Mammalian 15-lipoxygenases undergo a characteristic self-inactivation. The oxygenation of a single methionine to methionine sulfoxide, by 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE), was previously suggested as the cause of the inactivation of rabbit reticulocyte lipoxygenase. The site of oxygenation is potentially near the enzyme's active site; however, the specific location of the modified amino acid residue has not been identified. To determine which of the methionine residues is oxygenated, we inactivated both human and rabbit 15-lipoxygenases with 13-HPODE and sequentially denatured, reduced, carboxymethylated, and digested the enzymes with trypsin. The digested mixtures were analyzed by reverse-phase HPLC chromatography. Mass spectrometric analysis of each of the methionine-containing fractions enabled us to locate the peptide segments containing the oxidized methionine in both enzymes separately. Tandem electrospray mass spectrometry identified the oxidized methionine residues to be amino acid 590 in the human enzyme and 591 in the rabbit enzyme. To investigate the significance of this oxygenation, Met590 in human 15-lipoxygenase was substituted with leucine by site-directed mutagenesis. The mutant protein was inactivated by 13-HPODE, yet no oxygenated peptide or other modified peptide could be identified by HPLC-MS analysis. We also found that human 15-lipoxygenase was inactivated during arachidonate oxidation and by the reaction product 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), and no modified peptide was detected. Thus, methionine oxygenation is not essential for the inactivation of human 15-lipoxygenase. We suggest, however, that Met590 is an amino acid in the substrate binding pocket of human 15-lipoxygenase and interacts with the enzyme product 13-HPODE.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1989

Stimulus-specific production of cyclooxygenase and lipoxygenase metabolites of arachidonic acid by bovine alveolar macrophages.

Alveolar macrophages (AMs) are capable of producing a variety of inflammatory mediators including those derived from arachidonic acid, the prostaglandins (PGs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). Inflammation associated with release of arachidonate-derived mediators is a result of the combined actions of all of these mediators. Thus, it is critical to determine the entire spectrum of arachidonate-derived metabolites that AMs are capable of producing. In this study bovine AMs were prelabeled with [3H]arachidonic acid prior to stimulation with serum-treated zymosan, phorbol myristate acetate (PMA), or the calcium ionophore A23187. The total release of arachidonate metabolites into the culture media was measured by reverse-phase HPLC with on-line radiometric detection. All stimuli used induced production of metabolites of the cyclooxygenase pathway with thromboxane B2 and HHT being the major metabolites. Lesser amounts of PGF2 alpha, PGE2, and PGD2 were produced. Only stimulation with A23187 resulted in production of LTB4 and 5-HETE, products of the 5-lipoxygenase pathway. This latter result indicates that the two major pathways of arachidonate metabolism in AMs may be selectively stimulated. Such an effect could have important consequences in the development of pulmonary inflammation. Furthermore, the spectrum of arachidonic acid metabolites produced by bovine AMs closely resembles that of human AMs, in contrast to rodent AMs.

Animals↗
PubMed1988

Production of lipoxygenase metabolites of eicosapentaenoic acid by bovine alveolar macrophages in vitro.

Lipoxygenase metabolites of arachidonic acid (AA), the leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs) are potent proinflammatory mediators. Release of LTs and HETEs by bovine alveolar macrophages (BAMs) was measured by reverse-phase high performance liquid chromatography. LTB4 (1.1 +/- 0.2 ng/10(6) cells) and 5-HETE (2.2 +/- 0.2 ng/10(6) cells) were the major metabolites calcium ionophore A23187-stimulated BAMs produced from endogenous AA. The tritiated forms of these compounds and their precursor fatty acids were produced following incorporation of [3H]AA into the cells and stimulation by calcium ionophore A23187. Incorporation of an alternative substrate, [3H]eicosapentaenoic acid [( 3H]EPA) into BAMs incubated in parallel resulted in production of [3H]LTB5 and [3H]5-hydroxyeicosapentaenoic acid (5-HEPE). Equivalent amounts of [3H]AA and [3H]EPA and of [3H]LTB4 and homologous [3H]LTB5 were released. BAM produced significantly greater amounts of [3H]5-HEPE than [3H]5-HETE, however. These findings indicate that the BAM 5-lipoxygenase is capable of metabolizing EPA to LTB5 and 5-HEPE, with the production of 5-HEPE preferred over 5-HETE.

Animals↗
PubMed1988

Determination of microsomal lauric acid hydroxylase activity by HPLC with flow-through radiochemical quantitation.

An assay for the microsomal hydroxylation of lauric acid (LA), based on HPLC with flow-through radiochemical detection, has been developed. Conditions were optimized for resolution and quantitation of three microsomal metabolites of LA, one of which has not been reported previously as a metabolite of LA in mammalian microsomal incubations. These products, 12-(omega)-hydroxy-LA, 11-(omega-1)-hydroxy-LA, and a novel metabolite, 10-(omega-2)-hydroxy-LA, were isolated by HPLC and identified by gas chromatography/mass spectrometry. In the presence of NADPH, the formation of all three metabolites was linear with time and microsomal protein concentration. Hydrogen peroxide also supported the microsomal metabolism of LA, although the ratio of metabolites was substantially different than that produced by NADPH-supported microsomes. Several biochemical probes (metyrapone, alpha-naphthoflavone, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, and 10-undecynoic acid) were used to dissociate the three LA hydroxylase activities. These experiments suggest that the site-specific hydroxylation [omega-, (omega-1)-, (omega-2)-] of LA may be catalyzed by different isozymes of cytochrome P-450.

Animals↗
PubMed1987

Biochemical characterization of hepatic microsomal leukotriene B4 hydroxylases.

omega-Hydroxylation of leukotriene B4 (LTB4) has been reported in human and rodent polymorphonuclear leukocytes; preliminary information indicates that this metabolism is cytochrome P-450 dependent. Therefore, these studies were initiated to characterize the cytochrome P-450-dependent metabolism of LTB4 in other tissues. LTB4 was metabolized by rat hepatic microsomes to two products, 20-hydroxy(omega)-LTB4 and 19-hydroxy(omega-1)-LTB4. The formation of these metabolites was both oxygen and NADPH dependent indicating that a monooxygenase(s) was responsible for these reactions. The apparent Km and Vmax for LTB4 omega-hydroxylase were 40.28 microM and 1202 pmol/min/mg of protein, respectively. In contrast, the apparent Km and Vmax for LTB4 (omega-1)-hydroxylase were 61.52 microM and 73.50 pmol/min/mg of protein, respectively. Both LTB4 omega- and (omega-1)-hydroxylases were inhibited by metyrapone in a concentration-dependent fashion. However, SK&F 525A inhibited LTB4 (omega-1)- but not omega-hydroxylase. In contrast, alpha-naphthoflavone decreased LTB4 omega- but not (omega-1)-hydroxylase activities. The differences in the Km apparent for substrate as well as the differential inhibition by inhibitors of cytochrome P-450 suggest that the omega- and (omega-1)-hydroxylations of LTB4 in hepatic microsomes are mediated by different isozymes of P-450. Furthermore, several additional characteristics of LTB4 hydroxylases indicate that these isozymes of P-450 may be different from those which catalyze similar reactions on medium-chain fatty acids, such as laurate and prostaglandins.

Animals↗
PubMed1987

Quantitative liquid chromatographic determination of disulfide-containing peptide analogues of vasopressin with dual Hg/Au electrochemical detection.

Quantitative methodology was developed for the analysis of disulfide-containing peptide analogues of vasopressin in biologic media. The procedure employs sample clean-up by an ion-exchange solid-phase extraction cartridge, followed by high-performance liquid chromatography with electrochemical detection. The detector is a dual Hg/Au system operated in series in which the disulfide-containing peptides are first reduced at the upstream electrode and then detected as free thiols at the downstream electrode. The assay is linear in the range 2-100 ng/ml (approximately 2-100 pmol/ml) of urine with a lower limit of detection of 1 ng (approximately 1 pmol) on column. The method displayed general utility for a number of structural analogues of vasopressin.

Chromatography, Liquid↗
PubMed1987

Arachidonic and eicosapentaenoic acid metabolism in bovine neutrophils and platelets: effect of calcium ionophore.

Substitution of dietary fatty acids has potential for altering the inflammatory response. The purpose of the present study was to define the metabolites of arachidonic acid (AA) and eicosapentaenoic acid (EPA) secreted by bovine peripheral blood neutrophils and platelets. High performance liquid chromatography was used to characterize cyclooxygenase and lipoxygenase metabolites secreted in response to the calcium ionophore A23187. Cells were prelabelled with 3H-AA or 3H-EPA prior to challenge with the calcium ionophore. Bovine neutrophils secreted leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) as the major metabolites of AA, as well as the corresponding leukotriene B5 (LTB5) and 5-hydroxyeicosapentaenoic acid (5-HEPE) metabolites of EPA. Peptidoleukotrienes derived from 3H-AA or 3H-EPA were not detected under these conditions. The major tritiated metabolites secreted from bovine platelets were: thromboxane A2, measured as the stable metabolite thromboxane B2 (TXB2); hydroxyheptadecatrienoic acid (HHT) and 12-HETE derived from 3H-AA; and the omega-3 analogs TXB3 and 12-HEPE, derived from 3H-EPA. Preferred substrate specificities existed amongst the AA- and EPA-derived metabolites for the intermediary enzymes involved in the arachidonic acid cascade. These findings support the hypothesis that substitution of membrane-bound AA by EPA has potential for modulation of the host inflammatory response following cellular phospholipid mobilization.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

'Metabolic mapping' of drugs: rapid screening techniques for xenobiotic metabolites with m.s./m.s. techniques.

The development of tandem mass spectrometry (m.s./m.s.) techniques for the rapid structural elucidation and quantification of metabolites is described. With these techniques the mass spectrometer serves as both a separation device as well as a tool for structure elucidation. Ion formation by desorption ionization techniques and mass analysis with an appropriate combination of neutral loss and precursor ion scans can then be used to classify the number and type of primary metabolites and polar drug conjugates that are present in a complex sample matrix. The resulting information can be used to assess the overall biotransformation routes that are available to a drug or other xenobiotic substance.

Animals↗
PubMed1985

Quantitative analysis of an N-oxide metabolite by fast atom bombardment tandem mass spectrometry.

An assay for the N-oxide metabolite of a benzazepine drug by fast atom bombardment ionization with tandem mass spectrometric analysis on a triple quadrupole mass spectrometer has been developed and validated for urine and plasma samples. This methodology allows analysis of this metabolite directly in crude sample extracts, without the need for extensive chromatography or sample derivatization. Quantification was accomplished with the use of a stable isotope analog of the analyte as an internal standard, using the selected reaction monitoring mode of operation.

Benzazepines↗
PubMed1983

Mg-2,4-divinyl pheoporphyrin a5: the product of a reaction catalyzed in vitro by developing chloroplasts.

The major product of an aerobic reaction mixture containing developing chloroplasts, Mg-protoporphyrin IX, S-adenosylmethionine, and other cofactors was isolated and purified. Structural studies using nuclear magnetic resonance confirmed earlier reports, based on fluorescence and absorption spectra, that this compound is Mg-2,4-divinyl pheoporphyrin a5. The molecular weight determined by secondary-ion mass spectroscopy further confirmed the assigned structure. Absorption and fluorescence spectra indicate that this compound is identical to that reported previously by various workers in less-purified biological extracts. The nuclear magnetic resonance spectrum of the Mg-free base also supports the assigned structure.

Aerobiosis↗
PubMed1982

Direct analysis of steroid conjugates: the use of secondary ion mass spectrometry.

Data are presented on the mass spectrometry of intact steroid conjugates. The principal technique used was secondary ion mass spectrometry (SIMS) using a Cs+ ion beam for ionization, although comparable data were obtained by fast atom bombardment (FAB) using a Xeo beam. In both techniques the samples were analyzed in a liquid matrix (glycerol). Positive and negative ion spectra have been obtained, the latter being most useful for steroid sulfate and glucuronide analysis. The negative ion spectra are dominated by a pseudomolecular ion at m/z [M-H]- (M of free acid) and the lack of marked fragmentation permits mixtures of steroids to be resolved in a single spectrum, providing they differ in mass. Preliminary data on the separate analysis of individual components from urine and plasma of patients with assorted disorders of steroid synthesis and metabolism are presented. This technique shows great promise for the clinical analysis of steroid conjugates without the need for enzymic hydrolysis or chromatographic separation of individual steroids.

Glucuronates↗
PubMed1981

Carcinogen binding to DNA.

A key initiating event in the induction of neoplasia by a chemical carcinogen is believed to be the covalent reaction of the carcinogen with the DNA of the target cell. Most carcinogens are not biologically active as such, but require metabolic conversion to a chemically reactive form (ultimate carcinogen). Chemical carcinogens undergo an extremely complex set of metabolic reactions, leading for the most part to inactive detoxification products as well as reactive electrophilic species. Direct structural identification of the carcinogen-DNA adduct will (1) immediately confirm that the chemical is acting as a potential carcinogen under a given set of circumstances; and (2) directly identify those critical metabolic pathways which are involved in the metabolism of the chemical to a carcinogenic form rather than an inactive detoxification product. The direct structural identification of carcinogen-DNA adducts represents a formidable analytical challenge, since only picomolar quantities can be isolated. The advent of newer ionization techniques, such as field desorption and mass spectrometric-based separation techniques capable of handling mixtures, are proving to be essential for the characterization of such structures. Examples of nucleic acid-carcinogen adduct structure characterizations that have led to fundamental insights into the mechanisms of chemical carcinogenesis will be discussed, and future trends in mass spectrometry that have a direct bearing on these difficult problems will be pointed out.

Binding Sites↗
PubMed1981

A comparison of the carcinogen-DNA adducts formed in rat liver in vivo after administration of single or multiple doses of N-methyl-4-aminoazobenzene.

N-Methyl-4-aminoazobenzene (MAB) is believed to be metabolized in the liver to an electrophilic N-sulfonyloxy ester which binds covalently to cellular macromolecules, resulting in the induction of hepatic neoplasia. Previous in vivo studies in the rat detected only two hepatic MAB-DNA adducts, 3-(deoxyguanosin-N2-yl)-MAB(N2-dG) and N-(deoxyguanosin-8-yl)-MAB(C8-dG), which respectively accounted for 25% and 70% of the total MAB bound to DNA at 8 h after a single dose of the carcinogen. Subsequently, the C8-dG adduct was shown to be rapidly lost from the DNA while the N2-dG adduct was a persistent lesion. Since a single dose of MAB is not sufficient for complete carcinogenic activity, we sought to identify the MAB-DNA adducts present in rat liver after multiple oral doses of [3H]MAB. The MAB was administered by intubation at a level of 0.2 mmol/kg for 1, 3 or 4 doses and animals were sacrificed at 8 h after the last dose. Hepatic DNA was isolated by extraction and hydroxylapatite chromatography and was enzymatically hydrolyzed to MAB-mononucleoside adducts, which were quantitated by high pressure liquid chromatography (HPLC). After 3 doses, N2-dG, C8-dG, and an unknown adduct were detected. By 4 doses, these accounted for 51%, 25% and 23% of the total adducts. This data is consistent with rapid removal of the C8-dG derivative and the relative persistence of the N2-dG and the unknown adduct. The latter was shown to exhibit chromatographic and pH-dependent solvent partitioning properties that were identical to a product also present in DNA treated with the synthetic ultimate carcinogen, N-benzoyloxy-MAB. Analysis of this adduct by field desorption mass spectrometry (M+ = 460) and, after perdeuteromethylation, by electron impact mass spectrometry (M+ = 528; M-N(CH3)(CD3) = 481) indicated the structure to be a deoxyadenosin-N6-yl derivative substituted through an aromatic ring of MAB. Further analysis by 270 MHz 1H-NMR spectroscopy allowed complete assignment of the MAB and adenyl resonances and was uniquely consistent with a 3-(deoxyadenosin-N6-yl)-MAB structure. Since this persistent adduct is potentially mutagenic due to possible tautomeric equilibria between the N6-amino and N6-imino structures, it may represent an initiating lesion in MAB hepatocarcinogenesis.

Animals↗
PubMed1981

Formation of DNA adducts by the carcinogen N-hydroxy-2-naphthylamine.

The probable ultimate urinary bladder carcinogen, N-hydroxy-2-naphthylamine (N-OH-2-NA), reacted with nucleic acids and proteins under mildly acidic conditions (pH 5) to form covalently bound derivatives. The extent of reaction was in the order: Polyguanylic acid greater than DNA approximately protein greater than rRNA greater than tRNA greater than polyadenylic acid approximately polyuridylic acid greater than polycytidylic acid. At pH 7, appreciable reaction occurred only with protein. Enzymatic hydrolyses of the DNA, which contained 1.5 naphthyl residues/1,000 nucleotides, yielded 3 nucleoside-arylamine adducts. From chemical, UV, nuclear magnetic resonance, and mass spectrometric analyses, the adducts were identified as 1-(deoxyguanosin-N2-yl)-2-NA, 1-(deoxyadenosin-N6-yl)-2-NA, and a purine ring-opened derivative of N-(deoxyguanosin-8-yl)-2-NA, tentatively identified as 1-[5-(2-6-diamino-4-oxopyrimidinyl-N6-deoxyriboside)]-2-(2-naphthyl)urea. Preliminary experiments with a dog given [3H]2-NA suggested the presence of these adducts in vivo. The properties of adducts derived from N-OH-1-NA and N-OH-2-NA and their possible roles in the initiation of carcinogenesis are discussed.

2-Naphthylamine↗
PubMed1981

Identification of the DNA adducts formed in vitro from N-benzoyloxy-N-methyl-4-aminoazobenzene and in rat liver in vivo after administration of N-methyl-4-aminoazobenzene.

The synthetic model ultimate carcinogen N-benzoyloxy-N-methyl-4-aminoazobenzene reacted in vitro with either calf thymus or rat liver DNA to yield approximately 1 bound residue per 1,000 nucleotides. The DNA was enzymatically hydrolyzed and subjected to high-pressure liquid chromatographic analysis which indicated the presence of at least 6 N-methyl-4-aminoazobenzene (MAB) adducts. Two of the products cochromatographed with MAB-DNA adducts formed in rat liver in vivo following oral administration of the precarcinogen MAB. These two adducts were identified by UV, mass, and nuclear magnetic resonance spectroscopy as N-(deoxyguanosin-8-yl)- and 3-(deoxyguanosin-N(2)-yl)-MAB: the first adduct was initially the predominant product in vivo, ut it could not be detected 7 days after treatment, and the second remained at a constant level for 14 days and therefore appeared to be a persistent lesion.

Animals↗
PubMed1980

Characterization of DNA adducts of the carcinogen N-methyl-4-aminoazobenzene in vitro and in vivo.

Since the susceptibility of specific tissues to tumor formation has been correlated with the persistence of DNA-carcinogen adducts, the identity and persistence of DNA adducts formed from the hepatocarcinogen N-methyl-4-aminoazobenzene (MAB) has been determined. The synthetic ultimate carcinogen N-benzoyloxy-N-methyl-4-aminoazobenzene (N-BxO-MAB) was reacted in vitro with either calf thymus or rat liver DNA to yield approx. 1 bound residue per 1000 nucleotides. After enzymatic hydrolysis of the DNA and high pressure liquid chromatographic analysis, at least six MAB adducts were detected. Two of the products cochromatographed with MAB-DNA adducts formed in rat liver in vivo following oral administration of the precarcinogen MAB. These two adducts were identified by mass, UV and nuclear magnetic resonance (NMR) spectroscopy as N-(deoxyguanosin-8-yl)- and 3-(deoxyguanosin-N2-yl)-MAB. The former adduct was initially the predominant product in vivo, but it could not be detected 7 days following treatment. The latter adduct remained at a constant level for 14 days and therefore appears to be a persistent lesion.

Animals↗