Metabolism of diclofop-methyl (methyl-2-[4-(2',4'-dichlorophenoxy)phenoxy] propanoate) in cell suspensions of diploid wheat (Triticum monococcum)

The metabolism of the herbicide, diclofop-methyl (methyl-2-[4-(2′,4′-dichlorophenoxy)phenoxy]propanoate, in cell suspensions of resistant diploid wheat (Triticum monococcum L.) was determined 1, 8, and 24 h after treatment with ¹⁴C-diclofop-methyl. The ¹⁴C-labeled products were identified by thin layer chromatographic comparisons to appropriate standards. Eight hours after treatment with 5 μM diclofop-methyl in 0.8% acetone (neither of which were toxic to the cell suspensions) 87.2% (84.0% methanol soluble, 3.2% methanol insoluble) of the total ¹⁴C recovered (90.4%) was in the cells and 12.8% was in the medium. Major metabolites found in methanol extracts of the cells were diclofop (2-[4-(2′,4′-dichlorophenoxy)phenoxylpropionic acid), diclofop hydroxylated at an undetermined position on the 2,4-dichlorophenyl ring (ring-OH diclofop), and conjugates of ring-OH diclofop. Acid hydrolysis of the conjugated metabolite(s) yielded ring-OH diclofop and diclofop. Twenty-four hours after treatment 70–75% of the total ¹⁴C recovered was present as conjugated metabolites. With the exception of ring-OH diclofop, all metabolites present in the cells were also recovered from the medium. A metabolite found in low concentrations in the medium that yielded diclofop upon hydrolysis was identified as an ester conjugate. Toxic concentrations of diclofop-methyl (10 and 20μM) had no effect on the metabolism of the herbicide, although the rate of uptake was slower than for cells treated with 5 μM herbicide. The products of diclofop-methyl metabolism in cell suspensions of T. monococcum were compared to previous data from T. aestivum intact plant metabolism of diclofop-methyl.     

Determination of metabolites of pyrethroids in human urine using solid-phase extraction and gas chromatography–mass spectrometry

The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, λ-cyhalothrin, fenvalerate, phenothrin and β-cyfluthrin in human urine in one run. The major urinary metabolites of these substances are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl₂CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br₂CA), fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA). After acidic hydrolysis to release the conjugated carboxylic acid metabolites, the analytes were separated from the matrix by means of solid-phase extraction using a reversed-phase column. The components of the eluate were converted to their methyl esters and extracted in hexane. Separation and quantitative analysis of the pyrethroid metabolites was carried out by capillary gas chromatography and mass selective detection. 2-Phenoxybenzoic acid served as an internal standard. The detection limits lay between 0.3 and 0.5 μg per litre urine. The relative standard deviations of the within-series imprecision were between 1% and 6%. The relative recovery rates ranged between 90% and 98%. Using this method we determined the elimination of pyrethroid metabolites in 24-h urine samples from eight pest controllers after indoor application of permethrin. The detected concentrations ranged from 1 to 70 μg g⁻¹ creatinine.     

1-Methoxypyrene and 1,6-dimethoxypyrene: two novel metabolites in fungal metabolism of polycyclic aromatic hydrocarbons

The metabolism of pyrene by Penicillium glabrum strain TW 9424, a strain isolated from a site contaminated with polycyclic aromatic hydrocarbons (PAHs) was investigated in submerged cultures. The metabolites formed were identified as 1-hydroxypyrene, 1,6- and 1,8-dihydroxypyrene, 1,6- and 1,8-pyrenequinone, and 1-pyrenyl sulfate. In addition, two new metabolites were isolated and identified by UV, ¹H nuclear magnetic resonance, and mass spectroscopy as 1-methoxypyrene and 1,6-dimethoxypyrene. Experiments with [methyl-³H]S-adenosyl-l-methionine (SAM) revealed that SAM is the coenzyme that provides the methyl group for the methyltransferase involved. To our knowledge, this is the first time that methoxylated metabolites of PAHs have been isolated from fungal cultures. Received: 27 August 1996 / Accepted: 8 January 1997     

Metabolism of tritiated gibberellin a(20) in maize

After the application of 2.36 Curies per millimole [2,3-³H]gibberellin A₂₀ (GA₂₀) to 21-day-old maize (Zea mays L., hybrid CM7 × CM49) plants, etiolated maize seedlings, or maturing maize cobs, a number of ³H-metabolites were observed. The principal acidic (pH 3.0), ethyl acetate-soluble metabolite was identified as [³H]GA₁ on the basis of co-chromatography with standard [³H]GA₁ on SiO₂ partition, high resolution isocratic elution reverse phase C₁₈ high performance liquid chromatography and gas-liquid chromatography radiocounting. Two other acidic metabolites were identified similarly as [³H]GA₈ and C/D ring-rearranged [³H]GA₂₀, although gas-liquid chromatography radiocounting was not performed on these metabolites. Numerous acidic, butanol-soluble (e.g. ethyl acetate-insoluble) metabolites were observed with retention times on C₁₈ high performance liquid chromatography radiocounting similar to those of authentic glucosyl conjugates of GA₁ and GA₈, or with retention times where conjugates of GA₂₀ would be expected to elute. Conversion to [³H]GA₁ was greatest (23% of methanol extractable radioactivity) in 21-day-old maize plants. In etiolated maize seedlings, the C/D ring-rearranged [³H]GA₂₀-like metabolite was the major acidic product, while conversion to [³H]GA₁ was low.     

The Metabolism of the Herbicide Diphenamid (N-N-dimethyl-2,2-diphenyl-acetamide) in Cell Suspensions of Soybean (Glycine max)

The fate of the herbicide diphenamid was determined in cell suspensions of soybean [Glycine max (L.) Merr. ‘Wilkin’] at different stages of cell growth: early log phase (3 to 7 d), log phase (7 to 14 d), and stationary phase (14 to 18 d). [Carbonyl-¹⁴C]-diphenamid was added to the suspensions as an acetone solution. Neither diphenamid (2 to 3 μM) nor acetone (0.5% v/v) was phytotoxic. The ¹⁴C-labeled products were identified tentatively by thin layer chromatographic comparison with reference compounds. The major metabolic products formed were N-hydroxymethyl-N-methyl-2,2-diphenylacetamide, N-methyl-2,2-diphenylacetamide, 2,2-diphenylacetamide, and two polar metabolites (0.9 to 25% of the applied ¹⁴C activity) that appeared to be glucose conjugates; one an acidic glucoside. All metabolites were found in both the cell extract and the culture medium, except for the acidic glucoside, which was recovered in small amounts only from the cell extracts. These products were the same as those recovered from intact plants. Similar results were obtained from cell suspensions of different ages. The rate of metabolism by log phase cells was slightly less than the rate for either young or old cells. The results indicated that soybean cell suspensions can be used to obtain reliable information on the fate of agricultural chemicals in soybeans.     

Characterization of Fluoroglycofen Ethyl Degradation by Strain <Emphasis Type="Italic">Mycobacterium phocaicum</Emphasis> MBWY-1

A fluoroglycofen ethyl-degrading bacterium, MBWY-1, was isolated from the soil of an herbicide factory. This isolated strain was identified as Mycobacterium phocaicum based on analysis of its 16S rRNA gene sequence and its morphological, physiological, and biochemical properties. The strain was able to utilize fluoroglycofen ethyl as its sole source of carbon for growth and could degrade 100 mg l⁻¹ of fluoroglycofen ethyl to a non-detectable level within 72 h. The optimum temperature and pH for fluoroglycofen ethyl degradation by strain MBWY-1 were 30°C and 7.0, respectively. Five metabolites produced during the degradation of fluoroglycofen ethyl and were identified by mass spectrometry as {5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrophenylacyl} hydroxyacetic acid, acifluorfen, 5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrobenzoate, 5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-hydroxyl, and 3-chloro-4-hydroxyl benzotrifluoride. Identification of the metabolites allowed to propose the degradation pathway of fluoroglycofen ethyl by strain MBWY-1. The inoculation of strain MBWY-1 into soil treated with fluoroglycofen ethyl resulted in a higher fluoroglycofen ethyl degradation rate than in uninoculated soil regardless of whether the soil was sterilized or nonsterilized.     

Evaluation of lactic acid bacterium from chilli waste as a potential antifungal agent for wood products

Aims: The aim of this study was to isolate lactic acid bacteria from chilli waste and evaluate metabolites produced for the ability to arrest wood decay. Methods and Results: Using an optical density screening method, one bacterium (isolate C11) was identified as having pronounced antifungal properties against Oligoporus placenta. This isolate was identified as Lactobacillus brevis by 16S rRNA gene sequencing. To determine antifungal activity in wood, Pinus radiata blocks were impregnated with Lact. brevis [C11] cell‐free supernatant and exposed to brown rot fungi O. placenta, Antrodia xantha and Coniophora puteana. The treated timber demonstrated resistance to degradation from all fungi. The antifungal metabolites were heat stable and not affected by proteinase K, but were affected by neutralization with NaOH suggesting the metabolites were of an acidic nature. The presence of lactic and acetic acid was confirmed by HPLC analysis. Conclusions: Lactobacillus brevis [C11] produced acidic metabolites that were able to inhibit the growth of wood decay fungi and subsequent wood decay. Significance and Impact of the Study: Traditional wood treatments are becoming an environmental issue as the public demands more benign options. The use of lactic acid bacteria which are considered safe for general use is a potential alternative to the conventional heavy metal chemicals currently in use.     

THE RELEASE IN VIVO OF DOPAMINE SYNTHESIZED FROM LABELLED PRECURSORS IN THE CAUDATE NUCLEUS OF THE CAT

Abstract— To study the release of dopamine (DA) evoked in vivo from the caudate nucleus, a push-pull cannula was inserted into the head of the caudate nucleus of cats anaesthetised with pentobarbitone sodium (Nembutal), and the tissue in the vicinity of the cannula tip was continuously irrigated with either l -[¹⁴C]tyrosine or DL-[¹⁴C]3,4-dihydroxyphenylala-nine (DOPA). The contents of [¹⁴C]DA and of the [¹⁴C]acidic metabolites in the perfusates were determined after separation from the labelled precursors by column chromatography, TLC and solvent partition. During perfusion with radioactive tyrosine, only small quantities of [¹⁴C]DA appeared in the effluent while the concentrations of the [¹⁴C]acidic metabolites gradually increased during the course of the experiment. When [¹⁴C]DOPA was substituted for [¹⁴C]tyrosine, the proportion of precursor that was converted to DA and released into the effluent as the amine or as its acidic metabolites was increased ten-fold. In an attempt to increase the resting release of [¹⁴C]DA, D-amphetamine, tropolone or pheniprazine were individually added to the perfusion fluid. Each drug increased the content of [¹⁴C]DA in the perfusate, but the enhanced release was maintained only when pheniprazine was added during perfusion with [¹⁴C]DOPA. Stimulation of the rostral substantia nigra (A5-5) and the medial forebrain bundle caused, in a majority of experiments, a two-to five-fold increase in the concentration of labelled DA in the effluent. Stimulation of the substantia nigra at A4-0 did not enhance the release of [¹⁴C]DA from the caudate nucleus but did enhance the release from the putamen. Since the increase in the output of [¹⁴C]DA was independent of changes in the output of labelled acidic metabolites, the evoked release was apparently not attributable to changes in extracellular fluid dynamics.     

Analysis of 4-aminobiphenyl in smoker’s and nonsmoker’s urine by tandem mass spectrometry

The aromatic amine 4-aminobiphenyl (4-ABP) is present in tobacco smoke. In humans, it is also a known bladder carcinogen. We describe here a method for the quantification of total 4-ABP in urine using capillary gas chromatography/tandem mass spectrometry, with an effective detection limit in urine samples of approximately 0.87 pg/mL. We also examined the efficiency of chemical or enzymatic hydrolysis of urinary aromatic amine metabolites. Although we found acidic or basic hydrolysis effective, we found enzymatic hydrolysis (β-glucuronidase with either Escherichia coli or Helix pomatia) ineffective. As part of this work, we also confirm the presence of N-acetyl-4-ABP and 4-ABP glucuronide in human urine samples from smokers. These metabolites have been reported in animal studies, but previously they have not been identified in human samples. These metabolites, however, were found to be unstable and thus infeasible for biomonitoring. The final validated urinary total 4-ABP assay was applied to the analysis of samples from smokers and nonsmokers, whose status was confirmed from cotinine EIA measurements. Among 41 confirmed nonsmokers, the geometric mean (95% CI) of 4-ABP concentration was 1.64 pg/mg creatinine (1.30–2.07). Conversely, in 89 smokers, the geometric mean of 4-ABP concentration was significantly greater, at 8.69 pg/mg creatinine (7.43–10.16), p?<?0.001. Our results indicate that following tobacco smoke exposure, total urinary 4-ABP is a reliable biomarker for exposure to this carcinogen.     

Metabolism of DL-Tryptophan-3-14C by the Fruitlets of Citrus aurantifolia

Developing lime fruit [Citrus aurantifolia (Christm.) Swingle] was supplied with dl-tryptophan-3-¹⁴C in a special medium. An incubation period of six hours was sufficient for the radioactivity to reach an equilibrium between the fruit tissue and the incubation medium. Analyses of the fruit tissue and the medium for acidic and neutral metabolites of tryptophan indicated the existence of indolic products. The auxin indole-3-acetic acid (IAA) was identified among the products by dry column chromatography and biological assay. Other acidic metabolites included indolepyruvic acid and an unidentified material. Neutral metabolites included indolealdehyde, indoleacetaldehyde, and two unidentified compounds. Biological activity in the Avena curvature test was obtained from extracted compounds which corresponded to IAA and indolepyruvic acid in the acidic fraction and indoleacetaldehyde in the neutral fraction. Radioactive tryptophan was also found in both the acidic and the neutral fractions due to its amphoteric nature. The experiment demonstrated the conversion of tryptophan to its indolic metabolites, including indole-3-acetic acid, in this Citrus tissue.     

Formation of sulfate and glucoside conjugates of benzo[e]pyrene by Cunninghamella elegans

 Benzo[e]pyrene is a pentacyclic aromatic hydrocarbon, which, unlike its structural isomer benzo[a]pyrene, is not a potent carcinogen or mutagen. The metabolism of benzo[e]pyrene was studied using the filamentous fungus Cunninghamella elegans ATCC 36112. C. elegans metabolized 65% of the [9, 10, 11, 12-³H]benzo[e]pyrene and unlabeled benzo[e]pyrene added to Sabouraud dextrose broth cultures after 120 h of incubation. Three major metabolites of benzo[e]pyrene were separated by reversed-phase high-performance liquid chromatography. These metabolites were identified by ¹H and ¹³C NMR, UV-visible, and mass spectral analyses as 3-benzo[e]pyrenylsulfate, 10-hydroxy-3-benzo[e]pyrenyl sulfate, and benzo[e]pyrene 3-O-β-glucopyranoside. Received: 7 September 1995/Received revision: 14 November 1995/Accepted: 11 December 1995     

Structure of the O-polysaccharide of Providencia alcalifaciens O25 containing an amide of D-galacturonic acid with N(ε)-[(R)-1-carboxyethyl]-L-lysine

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O25 followed by gel-permeation and anion-exchange chromatography. The O-polysaccharide was studied by sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including two-dimensional correlation ¹H,¹³C HMBC, and ¹H,¹H ROESY experiments both in D₂O and, to detect correlations for NH protons, in a 9: 1 H₂O/D₂O mixture. An amino acid was isolated from the polysaccharide by acid hydrolysis and identified as N ^ɛ-[(R)-1-carboxyethyl]-L-lysine (“alaninolysine”, 2S,8R-alaLys) by determination of the specific optical rotation and ¹³C NMR spectroscopy, using the authentic synthetic diastereomers 2S,8R-alaLys and 2S,8S-alaLys for comparison. The structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established.     

Metabolism of auxin in pine tissues: Indole-3-acetic acid conjugation

Incubation of sections of various tissues of Pinus pinea L. with a relatively low concentration (3.6 μM) of indole-3-acetic acid-2-¹⁴C (IAA) resulted in the formation of two major metabolites. The first, which has not been identified, seemed to be a polar acidic compound and the second was identified as indole-3-acetylaspartic acid (IAAsp). The polar acidic metabolite has been found to be the major metabolite in needles, shoot wood and roots, while IAAsp has been found to be the major metabolite in shoot bark. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of a third metabolite which proved to be l-O-(indole-3-acetyl)-β-d -glucose (IAGlu) and a concomitant decrease in the amount of the polar acidic metabolite. This phenomenon was prominent particularly in needles. IAGlu was isolated from needles and IAAsp was isolated from shoot bark by means of polyvinylpolypyrrolidone column chromatography and preparative thin-layer chromatography. IAGlu was identified by comparison with authentic material by co-chromatography in three different solvent systems and by ¹H-nuclear magnetic resonance analysis. IAAsp was identified by comparison with authentic material by gas-liquid chromatography and ¹H-nuclear magnetic resonance analysis. Several aspects of formation, separation and isolation of IAA metabolites are discussed.     

Metabolism of [14C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots

Reverse-phase high-performance liquid chromatography was used to analyse ¹⁴C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [¹⁴C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [¹⁴C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [¹⁴C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid     

Metabolism of (±) 2-[14C]-abscisic acid in Lactuca sativa achenes

Achenes ofLactuca sativa L. cv. Grand Rapids were treated with (±) 2-[¹⁴C]-abscisic acid (ABA) at 10⁵ - or 2-10⁶ M for 6, 12, 24, 48 or 96 h in darkness at 24°C. They were then extracted in 80% ethanol. Two acidic diethyl ether phases which contained the free acids and the acids released after mild alkaline hydrolysis respectively, were analyzed as well as the radioactivity which remained in the final aqueous phase. For treatment durations between 6 and 96 h, the major part of the radioactivity was found in the free phase, in the form of ABA. For treatment durations up to 48 h, no radioactivity was detected at the Rf of phaseic acid or dihydrophaseic acid (free and hydrolysed phases). After 96 h culture on 10⁵ M ABA, dihydrophaseic acid was present, but only in very small quantities. Two ABA metabolites were detected. One was characterized as β-d -glucopyranosyl abscisate since its Rf was the same as that of an authentic sample in three different solvent systems and also since it released ABA on mild alkaline hydrolysis. It increased steadily with time and represented the main metabolite. The other metabolite found in the aqueous phase after mild alkaline hydrolysis and extraction with ether at pH 3 was a very polar compound, resistant to alkaline hydrolysis in the presence of concentrated ammonia and to methylation. It was, however, metabolized by apple embryo, yielding essentially dihydrophaseic acid and an ester which released dihydrophaseic acid on mild alkaline hydrolysis. These results indicate that under the conditions tried, the metabolism of [¹⁴C]-ABA by lettuce achenes leads almost exclusively to the formation of conjugates, oxidative metabolism of ABA being almost non-existent. Separate analysis of the integuments and of the endosperm plus embryo after culture of whole achenes for 48 h in the presence of 10⁵ M [¹⁴C]-ABA showed that ABA metabolism occurred only in the endospermembryo tissue.     

Characterization of a K+-ATPase from Lactobacillus helveticus ATCC 15009

Lactobacillus helveticus ATCC 15009 (wild-type) membrane preparations hydrolyzed Mg²⁺-ATP as a function of K⁺ concentration (2–200 mM). Mg²⁺-ATP hydrolysis by L. helveticus membranes was strongly inhibited in the absence of exogenous K⁺, while it amounted to 6 nmol ATP hydrolyzed min^–1 (mg membrane protein)^–1 at 50 mM KCl (saturating conditions) and pH 7.2. The K⁺-dependent ATPase of L. helveticus displayed a relatively high affinity for potassium ions (K _m = 800 μM) and was not affected by pretreatment of membranes with N,N’-dicyclohexylcarbodiimide. Membrane preparations were subjected to hypotonic shock to obtain a maximum yield of open profiles. The formation of a maximum level of enzyme-phosphate complex with a molecular mass of approximately 82 kDa was induced upon treatment of L. helveticus membrane preparations with low concentrations of [γ-³²P]ATP in the presence of K⁺ and La³⁺ ions and was visualized by acidic SDS-PAGE. It was concluded that L. helveticus membranes contain an inwardly directed K⁺ pump whose presence is discussed in terms of its putative role in cytoplasmic pH regulation. Received: 16 December 1996 / Accepted: 14 May 1997     

Identification by combined gas chromatography-mass spectrometry of phaseic acid and dihydrophaseic acid and characterization of further abscisic acid metabolites in pea seedlings

Seven day old seedlings of Pisum sativum L., cv. Kleine Rheinländerin, were wilted for 3 days. After partially removing the roots, they were rewatered and at the same time radioactive abscisic acid([1-¹⁴C]ABA, spec. activity 1.7·10⁸d s^-1mmol^-1) was applied for 1 h via the xylem of the roots. After 24 h, 4 days, and 12 days the seedlings were extracted and the metabolites of ABA were analyzed by means of thin-layer and gas chromatography in combination with mass spectrometry, autoradiography, and scintillation counting. Phaseic acid (PA) and dihydrophaseic acid (DPA) were identified as metabolites of ABA. The presence of another ABA-metabolite was also demonstrated. From its mass spectrum it has been postulated that this metabolite is 4-desoxy-ABA. In addition to these substances, several other metabolites, which are more polar than ABA and its known degradation products, were present in the seedlings. The quantity and number of these unknown metabolites increased with time.Abbreviations ABA abscisic acid - PA phaseic acid - DPA dihydrophaseic acid - TLC thin-layer chromatography - GC gas chromatography - PPO 2,5-diphenyloxazole - POPOP 2,2-p-phenylen bis(5-phenyloxazole)     

Gibberellin metabolism in excised lettuce hypocotyls: Evidence for the formation of gibberellin A1 glucosyl conjugates

The properties of the water-soluble metabolites of [³H]gibberellin A₁ ([³H]GA₁) from lettuce (Lactuca sativa L.) hypocotyls were compared with those of authentic samples of gibberellin (GA) glucosyl esters and ethers. Partitioning against l-butanol at high and low pH was not an efficient method of differentiating between ester and ether conjugates of GA₁ or GA₃. Extraction into l-butanol at pH 2.5 was, however, useful as a group purification step. Gel-filtration on acrylamide indicated a mean molecular weight of ca. 600 for the polar material and high-voltage electrophoresis separated two compounds (LH 1 and LH 2) with differing charge properties. Both metabolites incorporated ¹⁴C from glucose and ³H from GA₁. Subsequent enzymatic hydrolysis of LH 1 released material with identical properties to [¹⁴C]glucose together with a second uncharacterised component. Feeding with [³H]GA₁ methyl ester greatly reduced the formation of LH 1 but not LH 2. The metabolites were provisionally identified as GA₁-glucosyl ester (LH 1) and GA₁-glucosyl ether (LH 2).Abbreviations GA gibberellin - LH1 GA₃-glucosyl ester - LH2 GA₁-glucosyl ether - HVE high voltage paper electrophoresis - TLC thin-layer chromatography     

Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography–mass spectrometry

A sensitive negative ion chemical ionization (NCI) gas chromatographic–mass spectrometric (GC–MS) method was modified for the quantitation of valproic acid (VPA) metabolites generated from in vitro cDNA-expressed human microsomal cytochrome P450 incubations. The use of the inherent soft ionization nature of electron-capture NCI to achieve high sensitivity enabled us to conduct kinetic studies using small amounts of recombinant human P450 enzymes. The assay is based on the selective ion monitoring of the intense [M−181] fragments of pentafluorobenzyl (PFB) esters in the NCI mode, and has the following features: (1) a micro-extraction procedure to isolate VPA metabolites from small incubation volumes (100 μl); (2) a second step derivatization with tert.-butyldimethylsilylating reagents to enhance sensitivity for hydroxylated metabolites; (3) a short run-time (<30 min) while maintaining full separation of 15 VPA metabolites by using a narrow-bore non-polar DB-1 column plus a new temperature gradient; and (4) good reproducibility and accuracy (intra- and inter-assay RSDs <15%, bias <15%) by using seven deuterated derivatives of analytes as internal standards. The derivatives of mono- and diunsaturated metabolites, like the parent drug, produced abundant [M−181]⁻ ions while the hydroxylated metabolites gave an ion at m/z of 273, corresponding to the [M−181]⁻ ion of the tert.-butyldimethylsilyl ethers. In conclusion, the GC–NCI-MS analysis of valproate metabolites provided us with a high resolution and sensitivity necessary to conduct metabolic and kinetic studies of valproic acid in small volume samples typical of the in vitro cDNA-expressed micro-incubation enzymatic systems.     

Metabolism of [14C]cholesterol in Manduca sexta pupae: Isolation and identification of sterol sulfates,free ecdysteroids,and ecdysteroid acids

[¹⁴C]Cholesterol was injected into fifth-instar larvae of Manduca sexta, and the metabolites were isolated and identified from 8-day-old male and female pupae. A major portion of the metabolized cholesterol was esterified either with a sulfate group or with fatty acids. The predominant ecdysteroid metabolites were 20-hydroxyecdysone, 20,26-dihydroxyecdysone, 20-hydroxyecdysonoic acid, and 3-epi-20-hydroxyecdysonoic acid. Smaller amounts of ecdysteroids were identified as conjugates of 26-hydroxyecdysone, 3-epi-20-hydroxyecdysone, 20,26-dihydroxyecdysone, and its 3α-epimer. The metabolic profiles were similar for both male and female pupae. The two ecdysteroid acids were identified by nuclear magnetic resonance spectroscopy and chemical ionization mass spectrometry and by mass spectral analyses of their methyl esters. Detection of 3-epi-20-hydroxyecdysonoic acid as a major metabolite is significant, as its occurrence has been scarcely reported. 3-Epiecdysteroid acid formation is discussed as a possible ecdysteroid-inactivating pathway that may be operating specifically in lepidopterous insects or in particular developmental stages such as eggs or pupae.