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2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and “dilute and shoot” procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients.

Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.

Rat liver tissue homogenates were utilized for in vitro enzymatic conversion of 2,6-diethylaniline (DEA) to the important alachlor metabolite 4-amino-3,5-diethylphenyl sulfate (ADEPS), which was also generated as a radiolabeled standard for use in metabolism studies. ADEPS formation in rodents is associated with the production of other reactive metabolites implicated in alachlor rodent carcinogenesis, making dependable access to an ADEPS standard highly desirable. 14C-DEA was oxidized by rat liver microsomes to 14C-4-amino-3,5-diethylphenol, which was further converted to ADEPS via addition of the phosphosulfate transferase cofactor adenosine-3‘-phosphate-5‘-phosphosulfate. Microprobe NMR was used in conjunction with high-resolution mass spectrometry to fully characterize the resulting 14C-ADEPS and confirm its structure. Because microgram quantities sufficed for full characterization, the enzymatic transformation provides a viable alternative to radiosynthesis of 14C-ADEPS. Keywords: Alachlor; enyzmes; m...

In the gibberellin (GA) biosynthesis pathway, 20-oxidase catalyzes the oxidation and elimination of carbon-20 to give rise to C19-GAs. All bioactive GAs are C19-GAs. We have overexpressed a cDNA encoding 20-oxidase isolated from Arabidopsis seedlings in transgenic Arabidopsis plants. These transgenic plants display a phenotype that may be attributed to the overproduction of GA. The phenotype includes a longer hypocotyl, lighter-green leaves, increased stem elongation, earlier flowering, and decreased seed dormancy. However, the fertility of the transgenic plants is not affected. Increased levels of endogenous GA1, GA9, and GA20 were detected in seedlings of the transgenic line examined. GA4, which is thought to be the predominantly active GA in Arabidopsis, was not present at increased levels in this line. These results suggest that the overexpression of this 20-oxidase increases the levels of some endogenous GAs in transgenic seedlings, which causes the GA-overproduction phenotype.

A procedure for preparing 14C-labelled half-generation Starburst® PAMAM dendrimers is described. Controlled incorporation of radioisotope was achieved by Michael additions of amines with 14C-labelled methyl acrylate, resulting in dendrimers with specific activities between 30 and 45 mCi/ mmol in very high radiochemical purity.Copyright © 1998 John Wiley & Sons, Ltd.

Thiazopyr labeled with 13C/14C was found to be rapidly metabolized and eliminated by laying hens dosed orally for four consecutive days at either 1.3 or 10.4 mg/day. Over 90% of the total radioactive dose appeared in the excreta within 22 h after the final dose at both levels. Tissue residues were very low in the muscle and egg white (0.004−0.01 ppm) with somewhat higher levels observed for the liver, abdominal fat, skin with fat, egg yolk, and kidney (0.047−0.298 ppm). The most abundant tissue metabolite was a nitrile ester resulting from the breakdown of the thiazoline ring of thiazopyr. A carboxylic acid metabolite found in the liver and excreta was shown to arise from oxidation at C-3 of the isobutyl side chain of the nitrile ester. In vitro liver homogenate studies demonstrated the presence of similar metabolic pathways in hens, goats, and rats, although the levels of individual metabolites varied. Keywords: Thiazopyr; hens; metabolites; MS; NMR

Niemann-Pick type C (NPC) is a neurodegenerative lysosomal storage disorder caused by defects in the lysosomal proteins NPC1 or NPC2. NPC cells are characterized by reduced lysosomal calcium levels and impaired sphingosine transport from lysosomes. Natural killer (NK) cells kill virally infected/transformed cells via degranulation of lysosome-related organelles. Their trafficking from lymphoid tissues into the circulation is dependent on sphingosine-1-phosphate (S1P) gradients, sensed by S1P receptor 5 (S1P5). We hypothesized that NK-cell function and trafficking could be affected in NPC disease due to the combined effects of the lysosomal calcium defect and sphingosine storage. In an NPC1 mouse model, we found the frequency of NK cells was altered and phenocopied S1P5-deficient mice, consistent with defects in S1P levels. NK cells from NPC1 mice also had a defect in cytotoxicity due to a failure in degranulation of cytotoxic granules, which was associated with reduced lysosomal calcium levels. Affected NPC1 patients and NPC1 heterozygote carriers had reduced NK-cell numbers in their blood and showed similar phenotypic and developmental changes to those observed in the NPC1 mouse. These findings highlight the effects of lysosomal storage on the peripheral immune system.

Accurate mass analysis at high resolution can be used to determine elemental compositions and is often essential for conclusive metabolite identification. Reference standards can play an important role in the success of the analysis. We have discovered that perfluorinated acid anhydride/fluorinated alkylalcohol (PF-anh/F-alc) derivatives of glyphosate and aminomethylphosphonic acid are sensitively analysed by negative-ion electrospray ionization (ESI) and by positive-ion chemical ionization (CI). Based upon these results, PF-anh/F-alc derivatives of amino acids and PF-anh derivatives of primary amines were prepared, greatly expanding our collection of reference standards for accurate mass measurements. Sample ions are not suppressed since the reference standards can be selected based upon a narrow mass range of interest. They are volatile and, due to the degree of fluorination, have accurate masses favourably close to nominal mass. They are adequately soluble in reverse phase high-performance liquid chromatography solvents, making them particularly useful for ESI. Several examples of positive-ion CI and negative-ion electrospray MS accurate mass analyses of metabolites, obtained using the PF-anh/F-alc derivatives, are described. © 1997 John Wiley & Sons, Ltd.

Halosulfuron-methyl is the active ingredient in a new class of herbicide products by Monsanto which are used for the control of broadleaf and sedge weeds in corn, turf, sugarcane, and other crops. The metabolic basis for the corn and wheat selectivity of halosulfuron-methyl was studied using cell suspension cultures and young seedlings. Both seedlings and cell suspensions were found to completely metabolize halosulfuron-methyl within hours of incubation, suggesting that corn and wheat tolerance to halosulfuron-methyl was due to rapid metabolism or detoxification. In marked contrast, in a halosulfuron-methyl sensitive species such as soybean very little metabolism occurred. The key metabolic route by which corn rapidly inactivates halosulfuron-methyl was found to be hydroxylation of the pyrimidine ring followed by rapid conjugation with glucose. The predominant pathway in wheat was O-demethylation of the methoxy group of the pyrimidine ring. Further oxidative biotransformation of the early primary metabolites led to the cleavage of the pyrimidine ring to form pyrazolesulfonamide as a terminal metabolite.

Macrophages play a key role in host defense and in tissue repair after injury. Emerging evidence suggests that macrophage dysfunction in states of lipid excess can contribute to the development of insulin resistance and may underlie inflammatory complications of diabetes. Ceramides are sphingolipids that modulate a variety of cellular responses including cell death, autophagy, insulin signaling, and inflammation. In this study we investigated the intersection between TLR4-mediated inflammatory signaling and saturated fatty acids with regard to ceramide generation. Primary macrophages treated with lipopolysaccharide (LPS) did not produce C16 ceramide, whereas palmitate exposure led to a modest increase in this sphingolipid. Strikingly, the combination of LPS and palmitate led to a synergistic increase in C16 ceramide. This response occurred via cross-talk at the level of de novo ceramide synthesis in the ER. The synergistic response required TLR4 signaling via MyD88 and TIR-domain-containing adaptor-inducing interferon beta (TRIF), whereas palmitate-induced ceramide production occurred independent of these inflammatory molecules. This ceramide response augmented IL-1β and TNFα release, a process that may contribute to the enhanced inflammatory response in metabolic diseases characterized by dyslipidemia. Background: Ceramide is a critical lipid mediator of cellular stress responses relating to inflammation and insulin resistance. Results: Palmitate and LPS synergistically induce macrophage de novo ceramide biosynthesis. Conclusion: TLR4 signaling via TRIF and MyD88 in combination with palmitate up-regulates ceramide synthesis through a non-transcriptional mechanism. Significance: Understanding how lipid excess intersects with inflammatory signaling may provide insights into the pathogenesis of metabolic disease.

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

[ 13 C, 14 C]Triallate was administered orally to Sprague-Dawley rats. Twelve excreted metabolites were identified, the most abundant being 2,3,3-trichloro-2-propenesulfinic acid. Five metabolites derived from 2,3,3-trichloro-2-propenethiol were identified in excreta, including a methyl sulfone mercapturate whose structure was confirmed by X-ray crystallography. Three 2-chloroacrylate metabolites were identified, including a 2-chloroacrylate mercapturate whose structure was confirmed by X-ray crystallography

Rat liver enzyme preparations were used to investigate the mechanistic pathways of triallate metabolism. Incubation of [ 14 C]triallate with NADPH-fortified microsomes gave rise to metabolites generated via a number of reaction types, including sulfur oxidation/hydrolysis and hydroxylation of the allylic thiol moiety accompanied by rearrangement. Addition of glutathione (GSH) to microsomal incubations resulted in the formation of GSH conjugates arising via conjugate addition of glutathione to metabolic intermediates such as trichloroacrolein

Inducible, microsomal prostaglandin E synthase 1 (mPGES-1), the terminal enzyme in the prostaglandin (PG) biosynthetic pathway, constitutes a promising therapeutic target for the development of new anti-inflammatory drugs. To elucidate structure-function relationships and to enable structure-based design, an mPGES-1 homology model was developed using the three-dimensional structure of the closest homologue of the MAPEG family (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), mGST-1. The ensuing model of mPGES-1 is a homo-trimer, with each monomer consisting of four membrane-spanning segments. Extensive structure refinement revealed an inter-monomer salt bridge (K26-E77) as well as inter-helical interactions within each monomer, including polar hydrogen bonds (e.g. T78-R110-T129) and hydrophobic pi-stacking (F82-F103-F106), all contributing to the overall stability of the homo-trimer of mPGES-1. Catalytic co-factor glutathione (GSH) was docked into the mPGES-1 model by flexible optimization of both the ligand and the protein conformations, starting from the initial location ascertained from the mGST-1 structure. Possible binding site for the substrate, prostaglandin H(2) (PGH(2)), was identified by systematically probing the refined molecular structure of mPGES-1. A binding model was generated by induced fit docking of PGH(2) in the presence of GSH. The homology model prescribes three potential inhibitor binding sites per mPGES-1 trimer. This was further confirmed experimentally by equilibrium dialysis study which generated a binding stoichiometric ratio of approximately three inhibitor molecules to three mPGES-1 monomers. The structural model that we have derived could serve as a useful tool for structure-guided design of inhibitors for this emergently important therapeutic target.

Cytochrome P450-derived epoxyeicosatrienoic acids (EET) are biologically active metabolites of arachidonic acid that have potent effects on renal vascular reactivity and tubular ion transport and have been implicated in the control of blood pressure. EETs are hydrolyzed to their less active diols, dihydroxyeicosatrienoic acids (DHET), by the enzyme soluble epoxide hydrolase (sEH). 1,3-dicyclohexylurea (DCU), a potent sEH inhibitor, lowers systemic blood pressure in spontaneously hypertensive rats when dosed intraperitoneally. However, DCU has poor aqueous solubility, posing a challenge for in vivo oral delivery. To overcome this limitation, we formulated DCU in a nanosuspension using wet milling. Milling reduced particle size, increasing the total surface area by approximately 40-fold. In rats chronically infused with angiotensin II, the DCU nanosuspension administered orally twice daily for 4 days produced plasma exposures an order of magnitude greater than unmilled DCU and lowered blood pressure by nearly 30 mmHg. Consistent with the mechanism of sEH inhibition, DCU increased plasma 14,15-EET and decreased plasma 14,15-DHET levels. These data confirm the antihypertensive effect of sEH inhibition and demonstrate that greatly enhanced exposure of a low-solubility compound is achievable by oral delivery using a nanoparticle drug delivery system.

Introduction: Retinoid receptors are nuclear hormone receptors which are dynamically regulated during terminal myeloid maturation. Retinoic acid receptor α (RARA) is the target of at least ten fusion proteins that lead to acute promyelocytic leukemia (APL). All trans-retinoic acid (ATRA) has been thought to be the principle natural ligand for RARs and it has been used for the treatment of patients with APL. However, the enzymatic pathways that regulate natural retinoids metabolism in hematopoietic cells have not been well defined. ATRA is synthesized from vitamin A through two sequential steps. Vitamin A is oxidized by an alcohol dehydrogenase to yield retinal, which is then irreversibly oxidized by an aldehyde dehydrogenase (ALDH) to generate retinoic acid (RA). At least 19 different human ALDHs have been identified. Among them, ALDH1A1, ALDH1A2 and ALDH1A3 have been shown to oxidize all trans-retinal to ATRA with high affinity, which can be inhibited by diethylaminobenzaldehyde (DEAB). Whether other ALDHs participate in RA metabolism is unknown. Our study identified two distinct retinoid metabolism pathways that are active in bone marrow (BM) progenitors and in macrophages (Mφ). Methods: Gal4-UAS reporter system was used to detect natural RARA ligands in mouse primary hematopoietic cells. We transduced UAS-GFP mouse BM cells with retrovirus that expresses a fusion protein containing the DNA binding domain of Gal4 (which recognizes the UAS promoter) and the ligand binding domain of RARA. An IRES mCherry cassette was included for normalization purposes. Transduced cells were cultured in vitro, or transplanted into lethally irradiated recipient mice. Cells with intracellular RARA ligands activate GFP expression. GFP and mCherry expression were evaluated by flow cytometry. Real-time PCR and Affymetrix array were used to quantify ALDH expression. We identified RARA ligands by mass spectrometry (MS). Results: In vitro, we found that both mouse BM Kit+ cells (Kit+ progenitors) and BM-derived macrophages (BMMφ) could synthesize active RARA ligands via different pathways, but only when the cell culture media was supplemented with vitamin A. Kit+ progenitors utilize DEAB-sensitive ALDH pathways, whereas BMMφ use DEAB-insensitive pathways. By real-time PCR we found Kit+ progenitors have high expression of Aldh1a1, Aldh1a2 and Aldh1a3, whereas BMMφ have no detectable expression of these enzymes. We compared gene expression in Kit+ progenitors and BMMφ by Affymetrix profiling and found that Aldh3b1 was overexpressed in BMMφ. Ectopic expression of Aldh3b1 in 293T cells resulted activation of the same GFP reporter, which could be abrogated by two different antagonist, Ro41-5253 and BMS493, suggesting that Aldh3b1 generated an RARA specific ligand, which we subsequently identified as ATRA via MS. Reciprocally, we found that siRNA knock down of Aldh3b1 in BMMφ reduced the transactivation of the RARA-dependent GFP reporter. The X-RARA fusions have been proposed to act via dominant-negative mechanisms, decreasing retinoid-dependent transcription and myeloid maturation. Surprisingly, in vivo, only rare GFPdim cells were observed in BM cells and no GFP positive cells in peritoneal Mφ of UAS-GFP/Gal4-RARA transplant mice. As positive control, we treated mice with ATRA and observed a dose-dependent GFP increase in both cell types, suggesting that the in vivo reporter can respond to ATRA, but ATRA is not synthesized during adult hematopoiesis, and that dominant-negative inhibition of ATRA-dependent transcription may not be the predominant pathogenic effect of the X-RARA fusion oncoproteins. Conclusion: We have found that at least two distinct enzymatic pathways may be utilized in primary hematopoietic cells to synthesize active RARA ligands from vitamin A. Mouse BM Kit+ progenitors predominantly employ a set of DEAB-sensitive enzymes (Aldh1a1, Aldh1a2 and Aldh1a3), whereas Mφ utilize DEAB-insensitive pathways. We identified Aldh3b1 as a likely candidate and shown that it is capable of ATRA synthesis in vitro. Although these enzymes are expressed in primary BM cells, we found that this does not result in active intracellular RARA ligands in monocytes or Mφ in vivo, suggesting that the rate-limiting step in retinoid synthesis in vivo is likely to involve additional enzymes required for intracellular transport of protein-bound, serum-available vitamin A. Disclosures No relevant conflicts of interest to declare.

Cyclooxygenase 2 (COX-2) is an inducible enzyme involved in the production of prostaglandins and thromboxanes during inflammation. There are now several lines of evidence indicating that increased expression of COX-2 plays a functional role in the development and progression of malignant epithelial cancers. However, there is only limited data regarding the role of COX-2 in melanoma pathogenesis. In the present work, we retrospectively examined lesions through out the development of melanoma and metastatic disease (dysplastic nevi n = 10, melanoma in situ n = 4, stage II melanoma n = 10, stage III n = 4, stage IV n = 3, stage V n = 2, melanoma metastasis lymph nodes n = 13 metastasis to other sites n = 3). COX-2 was consistently observed in keratinocytes, dermal fibroblasts, and inflammatory cells in regions adjacent to benign evi and primary cutaneous melanomas. However, no COX-2 staining was detected in the nevi nor in the primary skin melanoma cells. In addition, COX-2 was undetected in all vertical and radial growth phase cases Interestingly, 13 out of 13 of the lymph node metastasis expressed extremely high levels of COX-2 in overlying epithelium and inflammatory cells, and COX-2 was strongly detected in the metastatic cancer cells per se. For additional information on the expression of COX-2 in malignant melanoma, we determined the expression of COX-2 protein in several different melanoma cell lines. We found that 3We found that 5 out of 7 of the melanoma cells over expressed COX-2 compared to normal melanocytes. Collectively, these data suggest that COX-2 may play a functional role in metastases of melanoma, and treatment with COX-2 inhibitors may be efficacious for malignant melanoma.

Glucocorticoids, acting through the glucocorticoid receptor, potently modulate immune function and are a mainstay of therapy for treatment of inflammatory conditions, autoimmune diseases, leukemias and lymphomas. Moreover, removal of systemic glucocorticoids, by adrenalectomy in animal models or adrenal insufficiency in humans, has shown that endogenous glucocorticoid production is required for regulation of physiologic immune responses. These effects have been attributed to suppression of cytokines, although the crucial cellular and molecular targets remain unknown. In addition, considerable controversy remains as to whether glucocorticoids are required for thymocyte development. To assess the role of the glucocorticoid receptor in immune system development and function, we generated T-cell-specific glucocorticoid receptor knockout mice. Here we show that the T-cell is a critical cellular target of glucocorticoid receptor signaling, as immune activation in these mice resulted in significant mortality. This lethal activation is rescued by cyclooxygenase-2 (COX-2) inhibition but not steroid administration or cytokine neutralization. These studies indicate that glucocorticoid receptor suppression of COX-2 is crucial for curtailing lethal immune activation, and suggest new therapeutic approaches for regulation of T-cell-mediated inflammatory diseases.

The metabolism, covalent binding and hepatotoxicity of butylated hydroxytoluene (BHT, 4-methyl-2,6-di-t-butylphenol) and two congeners (E-BHT, 4-ethyl-2,6-di-t-butylphenol; I-BHT, 4-isopropyl-2,6-di-t-butylphenol) were compared using precision-cut liver slices prepared from phenobarbital (PB)-treated male Sprague-Dawley rats. At equimolar concentrations (1 mM) BHT was the most toxic of the three compounds, causing an 80% decrease in cell viability over a 6 h incubation period. E-BHT was intermediate in toxicity while the isopropyl derivative was relatively nontoxic. Intracellular glutathione levels decreased prior to the onset of cytotoxicity. The cytochrome P450 inhibitor metyrapone completely inhibited the toxicity of all three compounds. The rates of metabolism of the three compounds to glutathione conjugates were compared in both PB-treated microsomes and PB-induced liver slices. In both models, the rate of formation was greatest for BHT, followed by E-BHT and I-BHT. Synthetic quinone methides (QMs) were prepared from each parent phenol and the rates of reactivity with three nucleophiles (water, methanol and glutathione) were compared. With each nucleophile, BHTQM was the most reactive, while I-BHTQM was the least reactive. Finally, covalent binding to protein was assessed in two ways. First, alkylation of an isolated model protein (bovine insulin) was measured in a microsomal enzyme activation system by mass spectrometry. Incubations with BHT produced the greatest extent of protein alkylation, followed by E-BHT, while no alkylation was observed with I-BHT. In the second system, covalent binding to cellular protein was assessed in rat liver PB microsomes and tissue slices by Western blotting using an antibody specific for the tert-butylphenol portion of the compounds. Binding was greatest for BHT, intermediate for E-BHT and could not be detected for I-BHT. The alkylation pattern for E-BHT was strikingly similar to that of BHT, suggesting that both compounds bound similar proteins. In summary, our results suggest that for hindered phenols such as BHT, increasing the length of the 4-alkyl substituent retards the rate of formation of reactive intermediates, significantly reduces the electrophilicity of the reactive intermediate, and greatly reduces the amount but not the selectivity of covalent binding to cellular protein, thereby reducing the toxicity of the parent compound.

This study demonstrates a useful application of on-line microbore high-performance liquid chromatography (HPLC) fast atom bombardment (FAB) and thermospray (TSP) mass spectrometry techniques for identification of metabolites from the in vitro metabolism of an experimental Monsanto herbicide: 2-chloro-N-(ethoxymethyl-N-[2-methyl-6- (trifluoromethyl)phenyl]acetamide, 'chloroacetanilide'. The microbore HPLC FAB technique on a high-resolution sector mass spectrometer accelerated identification of polar metabolites from the in vitro metabolism study of the herbicide. It provided good chromatographic resolution and excellent FAB sensitivity with strong protonated molecular ions. Scanning high-resolution LC/FAB mass spectrometry also provided molecular formulae for structural elucidation of unknown metabolites. Sample purification and concentration were minimized. Identification of less polar metabolites was carried out using LC/TSP mass spectrometry with a quadrupole mass spectrometer. LC/TSP mass spectrometry provided useful structural information for both polar and less polar metabolites because their spectra showed more fragmentation than FAB spectra. Glutathione conjugation was the major reaction observed during in vitro incubation of the herbicide. Oxidation of the chloroacetanilide by rat liver enzymes was also a significant metabolic reaction. Seven metabolites were identified, of which four were glutathione conjugates.

Cross-polarization magic-angle spinning (CPMAS) {sup 13}C NMR spectroscopy has been used to characterize covalent conjugates of alachlor, an {alpha}-chloroacetamide hapten, with glutathione (GSH) and bovine serum albumin (BSA). The solid-state NMR method demonstrates definitively the covalent nature of these conjugates and can also be used to characterize the sites of hapten attachment to proteins. Three different sites of alachlor binding are observed in the BSA system. Accurate quantitation of the amount of hapten covalently bound to GSH and BSA is reported. The solid-state {sup 13}C NMR technique can easily be generalized to study other small molecule/protein conjugates and can be used to assist the development and refinement of synthetic methods needed for the successful formation of such protein alkylation products.

Fast atom bombardment mass spectrometry (FAB/MS) is one of the most successful soft ionization methods for MS analysis of nonvolatile, thermolabile, polar organic compounds. The ionization occurs as a sputtering process caused by impact of the activated fast atom beam on the sample which is generally dissolved in a viscous liquid such as glycerol. The minimun amount of the sample to obtain a good spectrum is usually 0.1 to 1.0 μg and the mass spectrum lasts 10 to 30 minutes. The sophisticated mass spectral techniques such as MS/MS and HR/MS can be successfully adapted to the FAB ionization to analyze complicated organic mixtures as well as biological samples.The ionization source and the ionization process, types of liquid matrices and their roles, instruments, linked scan, HR/MS for FAB/MS and their applications for organic and biological research areas are discussed.

ヒ素系触媒上のイソブチレンの気相接触空気酸化によるメタアクロレインの合成反応を,反応温度410～510℃,接触時間1秒,イソブチレン濃度10%以上の原料で検討した。先ず数種の含ヒ素2成分系触媒を検討した結果,鉄-ヒ素系触媒が最も有効であるが,ヒ素成分の離脱によって活性が劣化することを認めた。そこでこれを抑制し,触媒活性を安定する第三成分として水酸化リチウムの添加を試み良好な成績が得られた。上記反応条件下で,リチウムの添加はヒ素に対し原子比で約1以上が望ましい。適正反応温度は約470℃ であるが,リチウム添加量の増減によって上下する。メタアクロレイン生成率および選択率と触媒中As5+濃度との間に顕著な相関関係が見られた。酸素変化率を100%以下に保ち,触媒組成と活性を安定に維持できる範囲内でできるだけ高いイソブチレン濃度の原料ガスを使用することによって,高いメタアクロレイン生成濃度が得られ,この場合As成分はほとんどAs5+の状態であった。原子比1:2:2.5で調製した鉄-リチウム-ヒ素触媒を用い,反応温度約470℃,イソブチレン対空気の混合比1:約7～8,接触時間1秒で酸素変化率90～100%,メタアクロレイン生成率26～28%(生成濃度約3.0～3.4%),選択率約56%の成績が得られ,触媒組成は上記原子比が1:2:2となって定常な活性を示していることが認められた。

A new mass spectral method has been developed and used to identify biologically important thiols. This method is based upon the use of C-14 labeled N-para-bromophenylmaleimide (BPM) to selectively derivatize thiols. The isotopic label facilitates the isolation and purification of trace quantities of the maleimide thiol adducts by high performance liquid chromatography (HPLC) with radioactivity detection. The use of the bromine atom in the thiol derivative greatly enhances the detectability of the parent and fragment ions containing the BPM moiety because of the generation of the characteristic N + 2 doublet ions. Several examples of the use of this method to identify non-volatile thiol peptides such as glutathione (GSH) and homoglutathione (hGSH) by fast atom bombardment mass spectrometry (FAB-MS) are described in this report.