Aqueous Metabolites Research Articles

Application of metabolite profiling to the identification of traits in a population of tomato introgression lines

Naturally occurring variation in wild species can be used to increase the genetic diversity of cultivated crops and improve agronomic value. Populations of introgression lines carrying wild species alleles afford an opportunity to identify traits associated with the introgressed regions, and facilitate characterization of the biochemistry and genetics underlying these phenotypes. Understanding plant metabolic pathways and the interactions between genes, phenotype, and environment is fundamental to functional genomics. Successful analysis of the complex network of plant metabolism requires analytical methods able to record information on as many metabolites as possible. Metabolite profiling is used to provide a snapshot of the metabolome in samples which differ in a known factor such as genetic background. Differences between the metabolite profiles can identify those metabolites/metabolic pathways affected by the introgression and allow genetic maps for metabolic alterations to be established. A Time-of-Flight Mass Spectrometry method is presented, with associated data reduction, used for profiling aqueous metabolites fom tomato. Analysis of ripe fruits of two tomato species, Lycopersicon esculentum and L. pennellii, showed differences in the amounts of many metabolites, including organic acids and sugars. Six introgression lines, L. pennellii introgressions within L. esculentum, were also examined and showed that Principal Component Analysis can reveal subtle differences in metabolism of the introgressed lines when compared to their parents.

Modulation of adult rat benzo(a)pyrene (BaP) metabolism and DNA adduct formation by neonatal diethylstilbestrol (DES) exposure

This study seeks to elucidate the role of diethylstilbestrol (DES), a synthetic estrogen on benzo(a)pyrene (BaP) metabolism in the male rat reproductive tissues. Offspring of timed-pregnant Sprague-Dawley rats were neonatally treated on days 2, 4, and 6 post-partum with 1.45 μmol/kg of DES. Ten weeks after birth, the adult rats were challenged with radiolabeled benzo(a)pyrene ( 3H BaP) (10 μmol/kg) and the rats were sacrificed 2 h after BaP exposure. Prostrate, testis, lung, liver, urine and feces samples were collected and extracted using a mixture of H 2O, MeOH and CHCl 3. The extracts were analyzed by reverse phase HPLC. The concentrations of BaP organic metabolites in DES rats were lower compared to controls (vehicle-treated rats). On the other hand, concentrations of aqueous metabolites were significantly increased in DES treated animals. The toxication to detoxication ratios were significantly decreased in DES rats compared to controls. This trend is also reflected in the decreased concentrations of BaP-DNA adducts in DES rats. Collectively these results suggest that DES is capable of modulating the metabolic pathway of BaP towards detoxification thereby preventing the manifestation of toxicity.

Biotransformation and genotoxicity of fluoranthene in the deposit‐feeding polychaete Capitella sp. I

Polycyclic aromatic hydrocarbons, such as fluoranthene (Flu), are of environmental concern because of their persistence, toxicity, and mutagenic properties. In this study we examined the genotoxicity of Flu to Capitella sp. I using the comet assay. We assessed patterns of DNA damage as a function of uptake route and as a function of exposure and depuration time and related levels of DNA damage to published information on Flu-metabolite formation. Exposure to approximately 30 microg Flu/g dry-weight sediment or 50 microg Flu/L seawater resulted in significant DNA damage. The degree of DNA damage was time dependent during both exposure and depuration, and although exposure route had no effect on the maximum degree of DNA damage occurring, it did influence the time course of damage. Levels of damage declined despite continued exposure to Flu, providing evidence for the induction of one or more DNA repair mechanisms. Comparison with Flu-metabolite profiles suggests that DNA damage is associated with the production of aqueous metabolites. The transitory nature of the DNA damage and repair process may contribute important insights into the mechanisms of toxicant effects at the molecular level but limits the usefulness of such endpoints as biomarkers of exposure or effect in ecotoxicological studies.

Biotransformation of dissolved and sediment‐bound fluoranthene in the polychaete, Capitella sp. I

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the marine environment and have physicochemical properties that make them likely to bioaccumulate. The main purpose of this study was to investigate fluoranthene (FLU) uptake, biotransformation and elimination in the deposit-feeding polychaete Capitella sp. I exposed via different routes. Individual worms were exposed to either dissolved FLU or to both dissolved and sediment-bound FLU. The 14C-FLU was used as a tracer to determine the amounts of unmetabolized FLU (parent), water-soluble (aqueous)- and water-insoluble (polar) FLU metabolites and FLU residues (unextractable fraction) in sediment, water, and worm tissue. Capitella was capable of accumulating and biotransforming FLU regardless of route of exposure, thus suggesting that biotransformation activity is not restricted to gut tissues. Although both feeding and nonfeeding worms were able to biotransform FLU extensively, feeding worms eliminated/transformed parent FLU faster than both aqueous and polar FLU metabolites after transfer to clean conditions, whereas nonfeeding worms barely excreted FLU in any form. The high biotransformation capability of Capitella sp. I. along with the dependency of elimination on exposure route, demonstrates the potential importance of these processes in controlling the fate and effects of PAH and possibly other organic toxicants in contaminated marine sediments.

Synchronous fluorescence spectrometry of 1-hydroxypyrene: a rapid screening method for identification of PAH exposure in tissue from marine polychaetes.

The uptake of polycyclic aromatic hydrocarbons (PAHs) by marine deposit-feeding invertebrates can be determined by screening for PAH-derived metabolites. We identified 1-hydroxypyrene as the only intermediate metabolite in tissue of four species of deposit-feeding polychaetes, Nereis diversicolor, Nereis virens, Arenicola marina, and Capitella sp. I exposed to pyrene spiked sediment. Synchronous fluorescence spectroscopy (SFS) provides a fast and simple method for both qualitative and quantitative analysis of 1-hydroxypyrene in all four species. The SFS assay was validated using HPLC with ultraviolet detection. A good correlation between 1-hydroxypyrene concentrations determined by the two methods was observed. We used HPLC with fluorescence detection combined with enzymatic hydrolysis of conjugated metabolites to investigate species specific metabolite patterns. A tentative aqueous metabolite identification scheme indicates that Nereid polychaetes predominately make use of glucuronide conjugation whereas Capitella sp. I. and Arenicola marina appear to utilize predominantly sulfate and/or glucoside conjugation. The usefulness of 1-hydroxypyrene as a biomarker for PAH exposure in deposit-feeding invertebrates is discussed.

1‐hydroxypyrene glucuronide as the major aqueous pyrene metabolite in tissue and gut fluid from the marine deposit‐feeding polychaete Nereis diversicolor

Both 1-hydroxypyrene and 1-hydroxypyrene glucuronide are identified as the primary phase I and phase II metabolites of the four-ringed polycyclic aromatic hydrocarbon (PAH) pyrene in the marine deposit-feeding polychaete Nereis diversicolor. Identification of pyrene and primary metabolites was performed using high-pressure liquid chromatography (HPLC) with diode-array detection and fluorescence detection (HPLC/DAD/F) and an ion-trap mass spectrometer for positive identification of 1-hydroxypyrene glucuronide. Besides 1-hydroxypyrene and 1-hydroxypyrene glucuronide, the HPLC/F trace of tissue samples from pyrene-exposed worms showed three additional low-intensity peaks that may be related to pyrene metabolism based on similar excitation/emission wavelengths. The peaks were all too low in intensity to be positively identified. Of the total PAH in tissue, 1-hydroxypyrene glucuronide, 1-hydroxypyrene, and pyrene constituted 73%, 2%, and 25% respectively. Gut elimination of metabolic products is supported by the identification of 1-hydroxypyrene and 1-hydroxypyrene glucuronide in both gut fluid and defecation water. Being the only phase I metabolite of pyrene, 1-hydroxypyrene becomes a useful marker for PAH exposure, and it may serve as a valuable model compound for assessing species-specific PAH metabolic capabilities.

Identification of 1-hydroxypyrene glucuronide in tissue of marine polychaete Nereis diversicolor by liquid chromatography/ion trap multiple mass spectrometry.

1-Hydroxypyrene glucuronide is identified as the single major aqueous metabolite of the tetracyclic aromatic hydrocarbon pyrene, in tissue from a deposit-feeding marine polychaete, Nereis diversicolor. Identification was performed using an ion trap mass spectrometer fitted with an atmospheric pressure chemical ionization (APCI) probe and connected to a high-performance liquid chromatography/diode array detector (HPLC/DAD) system. Besides 1-hydroxypyrene, the 339-nm UV trace of tissue samples from pyrene-exposed worms showed only one dominant peak that could be related to pyrene metabolism. Negative APCI-MS of this supposed 1- hydroxypyrene conjugate gave a characteristic signal at m/z 429 corresponding to the molecular ion of 1-hydroxypyrene glucuronide plus eluent adducts ([M - H + 2H(2)O](-)). Fragmentation pathways were studied by isolating the abundant ion at m/z 429 in the ion trap and performing multiple mass spectrometric experiments (MS(n)). The fragmentations observed were consistent with the proposed identification. Two low intensity LC peaks that could be related to pyrene metabolism by their DAD absorption spectra were also present in the 339-nm UV chromatogram of tissue samples. However, these peaks could not be identified by their mass spectra in negative ion mode due to ion suppression by very abundant co-eluting impurities. The present method shows that LC/MS(n) is a fast and useful analytical tool for identification of aqueous polycyclic aromatic hydrocarbon biotransformation products in samples from relatively small marine invertebrates with limited sample preparation.

The effects of paramagnetic contrast agents on metabolite protons in aqueous solution

The longitudinal (R1) and transverse (R2) relaxivities of the clinically used contrast agents Gd(DTPA)2−, Gd(DOTA)− and Gd(DTPA-BMA) have been determined in mixed aqueous metabolite solutions for choline, creatine and N-acetylaspartate. Measurements were performed at 1.5 T using a STEAM sequence on 25 mM metabolite solutions at pH = 7.4 and 22 °C. The data showed that for all the contrast agents and metabolites, R1 ∼ R2. The largest range of relaxivity values was found for Gd(DTPA)2−, where R2 = 6.8 ± 0.3 mM−1 s−1 for choline and 1.5 ± 0.4 mM−1 s−1 for N-acetylaspartate. Variation in relaxivity values was attributed primarily to differences between the charges of the paramagnetic agent and metabolite. The maximum potential influence of the contrast agents on in vivo metabolite signals was calculated using the measured relaxivities.

Metabolism, bioavailability, and toxicokinetics of Benzo(α)pyrenein F-344 rats following oral administration

The objective of this study was to evaluate the bioavailability of Benzo(a)pyrene [B(a)p], subsequent to oral exposure. Eight-week-old F-344 rats were dosed orally with 100 mg/kg body weight B(a)p and sacrificed at 0, 0.5, 1.0, 2.0, 4.0. 8.0, 24, 48 and 72 hours post exposure. Blood, liver, reproductive tissues, urine and fecal samples were collected at necropsy and were analyzed for parent B(a)p and metabolites by HPLC with fluorescence detection. Peak levels of B(a)p in plasma occurred 8 hours after exposure (67%) followed by a gradual decrease. Liver retained 10% of the administered B(a)p up to 24 hours following, which the levels dropped during the remaining time periods studied. Twenty-four hours after administration, 45% of the dose was excreted in feces and urine. Metabolite levels in plasma peaked at 24 hours (10%) and decreased to 1% at 72 hours. In the liver, metabolite levels were higher at 8 hours (10%) but were only 3% at 72 hours. Benzo(a)pyrene levels increased after 24 hours in the reproductive organs and constituted 10% of the administered dose at 72 hours. Blood showed high levels of 7,8-diol than 9,10 and 4,5-diols which were high in liver and reproductive organs. Compared to diols, the hydroxy metabolites were detected at high levels in urine and fecal samples. Among the aqueous phase metabolites, glucuronides were at higher levels compared to glutathiones and sulfates. The slow release of unmetabolized B(a)p from reproductive organs and the presence of reactive metabolites in these organs is a matter of concern as they could interfere with gonadal steroid synthesis and release and its regulatory role in gamete production, maturation and function of male animals in a continuous exposure paradigm.

Non-HPLC separation of water-soluble choline metabolites by two-dimensional high voltage electrophoresis and thin layer chromatography

In cholinergic neurons choline is directed to three main pathways; (1) conversion to phosphorylcholine (PCh) and cytidine diphosphate choline (CDP-choline) for the synthesis of phosphatidylcholine, (2) acylation to the neurotransmitter acetylcholine and (3) oxidation to betaine for the formation of methionine. Thus, the distribution of choline among the different metabolites is important for a better understanding of the regulation of these pathways in neurons. A non-HPLC method for the simultaneous separation of five choline metabolites found in neurons is described. High voltage electrophoresis (HVE) was combined with thin layer chromatography (TLC) to separate choline, PCh, CDP-choline, acetylcholine and betaine. This method is useful in studying the distribution of choline among its different metabolites in radiotracer experiments. Aqueous metabolites from leukemia inhibitory factor treated LA-N-2 cells labeled with [methyl- 3H]choline were separated by HVE followed by TLC in the same dimension. Although the separation appeared to be complete, some ‘tailing’ by PCh significantly elevated the radioactivity measured in CDP-choline. This tailing of PCh was confirmed by subjecting radiolabeled PCh alone to this multiple separation method. Contamination of CDP-choline by PCh was eliminated by subjecting the samples to HVE followed by TLC in the second dimension. This two-dimensional approach was consistently reproducible and achieved excellent resolution of all five metabolites. In addition, this technique also resolved a sixth choline-containing metabolite, glycerophosphorylcholine (GPC), a breakdown product of phosphatidylcholine.

Sources of variability in the response of coupled spins to the PRESS sequence and their potential impact on metabolite quantification.

Using a numerical method of solving the equation of motion of the density matrix, an evaluation is presented of the sources of the marked variability in the response to the point resolved spectroscopy (PRESS) pulse sequence of coupled proton spin systems. The consequences of an inappropriate 180 degrees pulse design and of the limitations on radiofrequency power are demonstrated for a weakly coupled example, lactate. The dominating role of strong coupling, which is present in most brain metabolites, is demonstrated for glutamate, in which 160 terms in the density operator were tracked to monitor the gross changes in lineshape and signal intensity as a function of the two echo times. The predictions of the numerical solutions were confirmed by experiments on phantoms of aqueous metabolite solutions.

Interaction of cadmium and benzo[a]pyrene in mummichog ( Fundulus heteroclitus): Biotransformation in isolated hepatocytes

Exposure of mummichog ( Fundulus heteroclitus), a common estuarine fish species, to simultaneous doses of cadmium (Cd) and benzo[a]pyrene (BP) has been shown to alter the acute toxicity of BP. In this study the effects of in vivo exposure to Cd and BP on the biotransformation of BP by isolated hepatocytes were investigated. Fish were injected intraperitoneally with sublethal doses of BP and Cd, and hepatocytes from these fish were isolated on days 3, 7 and 14. Hepatocytes were then co-incubated with radiolabeled BP. BP breakdown, and the formation of aqueous and organic solvent-extractable metabolites were quantified using HPLC. After three days there was a significant increase in metabolism in the BP exposed fish. The same increase was still noticeable on day 7, but started to return to baseline values on day 14. In the combined BP/Cd treated fish, BP metabolism was not significantly induced on day 3, but full induction was reached on day 7, and was still at this level on day 14. Analysis of the nonconjugated metabolites showed that hepatocytes produced predominantly BP-9,10-diol, BP-7,8-diol, BP-4,5-diol, and 9-hydroxy-BP. The results of this study suggest that Cd does not influence the profile of generated BP metabolites. However, the initial rate of BP breakdown is significantly decreased by pre-exposure to cadmium.

Formation of catechol estrogen glutathione conjugates and gamma-glutamyl transpeptidase-dependent nephrotoxicity of 17beta-estradiol in the golden Syrian hamster.

In an animal model of hormone-mediated carcinogenesis, male golden Syrian hamsters develop renal carcinoma following prolonged exposure to 17beta-estradiol. The basis for the species and tissue specificity is unclear. Detailed information on the disposition of 17beta-estradiol in this model is lacking. Because catechol estrogens have been implicated in this model of carcinogenesis, we investigated the metabolism and nephrotoxicity of 17beta-estradiol in golden Syrian hamsters, with emphasis on the formation of catechol estrogen thioethers. 17beta-Estradiol (50 micromol/kg, i.p.) is a mild nephrotoxicant, causing significant elevations in the urinary excretion of gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase, glutathione S-transferase (GST) and glucose. Increases in renal protein carbonyls and lipid hydroperoxides, which are markers of oxidative damage, also occur after administration of 17beta-estradiol (50 micromol/kg, i.p.). 17beta-Estradiol-mediated nephrotoxicity is reduced by treating animals with acivicin, an inhibitor of gamma-GT, implying that toxicity is mediated by metabolites requiring metabolism by this enzyme. Following administration of 17beta-[14C]estradiol (100 micromol/kg) to hamsters, 9.7% of the dose is recovered in bile after 5 h, the majority (7.9%) representing aqueous metabolites. Seven catechol estrogen GSH conjugates were identified, 2-hydroxy-1,4-bis-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-estrone, 2-hydroxy-1-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-17beta-estradiol, and 2-hydroxy-1-(glutathion-S-yl)-17beta-estradiol. At 5.4 micromol/kg of 17beta-estradiol, a dose-reflective of daily exposure levels in the hamster model of nephrocarcinogenicity, 12% of the dose is recovered within 5 h as a combination of GSH conjugates of 2- and 4-hydroxy-17beta-estradiol and 2- and 4-hydroxyestrone. In summary, oxidation of catechol estrogens, followed by GSH conjugation, occurs in vivo and 17beta-estradiol is a mild nephrotoxicant in a manner dependent on the activity of gamma-GT.

NADPH-dependent oxidation of benzidine by rat liver.

This study used liver microsomes from control an naphthoflavone-treated rats to evaluate NADPH-dependent oxidation of benzidine. With microsomes from beta-naphthoflavone-treated rats, the rates of formation of aqueous soluble metabolite (HPLC analysis) and protein and DNA binding were 835 +/- 81, 14.5 +/- 1.8 and 0.71 +/- 0.14 pmol/mg/min respectively. beta-Naphthoflavone treatment elicited 12.3-, 1.8- and 14.2-fold increases in benzidine metabolism compared with controls as judged by HPLC and protein and DNA binding respectively. For microsomes from treated animals, Km and Vmax values were 47 +/- 6 micromol and 1.13 +/- 0.16 nmol/mg protein/min respectively. All of the metabolic parameters were inhibited to varying degrees by glutathione (1 or 10 mM), N-acetylmethionine (10 mM) and ascorbic acid (10 mM). Following glutathione addition, at least two new metabolite peaks were observed, representing -6% of the total radioactivity recovered by HPLC. Neither metabolite was 3-(glutathion-S-yl)benzidine. Cytochrome P450 inhibitors (10 micro) specific for different members of cytochrome gene families 1-3 indicated that benzidine was metabolized by cytochrome P450 1A1/1A2. Ellipticine and alpha-naphthoflavone, specific 1A1/1A2 inhibitors, elicited 50% inhibition at -0.2 and 0.5 micro respectively. Electron impact and negative ion chemical ionization mass spectro- metry identified the aqueous soluble metabolite as 3-hydroxybenzidine. The lability of 3-hydroxybenzidine observed at pH > 7.0 was prevented by ascorbic acid. Thus, cytochrome P450 1A1/1A2 NADPH-dependent metabolism of benzidine to 3-hydroxybenzidine was demonstrated.

HMG-CoA reductase inhibitor and cholestyramine induce hepatic LDL receptor and are effective for lowering plasma cholesterol in homozygous Watanabe heritable hyperlipidemic rabbits

Male Fisher 344 rats were fed diets containing either 20% corn oil, 20% coconut oil or 18% cocomnut oil plus 2% corn oil for 3 wk. A single dose of [3H]aflatoxin B1 ([3H]AFB1) was administered ip and the biliary excretion of afflatoxin metabolites and the binding of 3H to nucleic acids were studied. In other experiments the in vitro metabolism of AFB1 by liver postmitochondrol supernatants prepared from rats fed the different sources of dietry fat was determined. The major nonextractable aqueous metabolite of AFB1 was the aflatoxin B1-glutathione conjugate (AFB1-GSH). Variation in the source of dietry fat did not affect production of the conjugate, nor was in vivo binding of AFB1 to nucleic acids affected. Aflatoxin P1 (AFP1)—mostly conjugated—and aflatoxin M1 (AFM1) were also identified in the bile, and the quantities of these metabolites produced were unaffected by the dietary treatments. The metabolites recovered in the in vitro study included aflatoxins Q1, P1 and M1. The corn oil diet produced a higher microsomal cytochrome P-450 level than the coconut oil diet. Associated with the higher level of cytochrome P-450 was increased in vitro coversion of AFB1 and AFM1, but not ot AFP1. The in vitro production of aqueous metabolites and the covalent binding of metabolites to protein was unaffected by the dietary treatments. The results of the in vivo studies suggest that the formation of the putative carcinogenic metabolite, AFB1-epoxide, which undergoes detoxification through glutathione conjugation, is not affected by the type of dietary fat. These observations further suggest that dietary fat, in particular unsaturated fat, affects AFB1 carcinogenisis through a mechanism other than by alteration of the metabolic activaqtion of AFB1.

High-Resolution Proton NMR Studies of Lymphocyte Extracts

Anatomic imaging is now a well-developed application of magnetic resonance. Greater capabilities for physiologic characterization should become possible by concomitant application of spectroscopic methods. High-resolution in vitro spectroscopy must first provide a framework upon which in vivo and diagnostic interpretation may be based. Biochemical profiles consisting of quantitation of extracted aqueous metabolites and lipids of particular cells or organs establish an in vitro glossary for what may be found in the intact cell or living subject. A large variety of amino acids, intermediary metabolites, membrane precursors, and nucleotides are detectable in extracts of human peripheral blood lymphocytes, and significant changes in intracellular concentrations have been monitored after lectin-induced activation. Corresponding changes in lipid profile have also been noted. An increasing variety of other cells and tissues are being similarly characterized. Despite its limitations, NMR analysis possesses the unique prospect of providing a noninvasive and nondestructive source of biochemical information.

Involvement of the lysosome in the catabolism of intracellular lysophosphatidylcholine and evidence for distinct pools of lysophosphatidylcholine.

The role of the lysosome in the metabolism of lysophosphatidylcholine was investigated in isolated rat hepatocytes. Chloroquine, primaquine, and ammonium chloride caused a 2.5-fold increase in radioactive lysophosphatidylcholine in [methyl-3H]choline-labeled cells. This effect was confirmed by a 1.7-fold increase in lysophosphatidylcholine mass in chloroquine-treated hepatocytes. Chloroquine caused a 2.7-fold increase in radioactive lysophosphatidylethanolamine in [1-3H]ethanolamine-labeled cells and a 2.3-fold increase in radioactive lysophosphatidylcholine in [methyl-3H]methionine-labeled cells. Chloroquine did not affect formation of choline-containing aqueous metabolites or the level of radioactivity in phosphatidylcholine (PC). The effect of chloroquine on radioactive lysophosphatidylcholine accumulation was concentration-dependent and occurred within 10 min, consistent with rapid inhibition of lysosomal function. As there was no observed decrease in the 3H in PC, the accumulation of lysophosphatidylcholine was likely due to the inhibition of acid lysophospholipase activity in chloroquine-treated cells. The accumulation of lysophosphatidylcholine in the presence of chloroquine was observed in both short-term- (30 min) and equilibrium-(24 h) [methyl-3H]choline-labeled cells. Simultaneous incubation of hepatocytes with both albumin and chloroquine increased the radioactivity in lysophosphatidylcholine in the medium independently of the accumulation of radioactive lysophosphatidylcholine in the cells. The results suggest that there are separate pools of lysophosphatidylcholine in the hepatocyte and that the pool donated to an extracellular acceptor is different from the lysosomal pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Contrasting Effects of 1,1,1-Trichloroethane on [14C] Vinyl Chloride Metabolism and Activation in Hepatic Microsomes from Phenobarbital- and Isoniazid-Treated Rats

The interaction of 1,1,1-trichloroethane (TCE). a widely used chlorocarbon solvent, on the metabolism and activation of [14C]-vinyl chloride in rat hepatic microsomes was investigated to determine the effects of combined exposures to these compounds. In microsomes from phenobarbital (PB)-treated rats, ICE increased vinyl chloride-protein binding and vinyl chloride aqueous metabolite formation over twofold when vinyl chloride 0.32% (v/v) and TCE (0.65%) are incubated together. In contrast, under similar incubation conditions, TCE inhibited vinyl chloride metabolism and protein binding up to 45% in microsomes from isoniazid-treated animals. TCE also inhibited vinyl chloride metabolism and binding in microsomes from untreated rats, but to a lesser degree. Like the effect of TCE on vinyl chloride-protein binding. TCE increased vinyl chloride binding to DNA approximately 130% in microsomes from PB-treated rats, yet inhibited vinyl chloride-DNA binding in microsomes from isoniazid-treated and untreated animals. Consistent with TCE effects on vinyl chloride binding and aqueous metabolite production, TCE further increased cytochrome P450 loss due to vinyl chloride metabolism in microsomes from PB-treated rats, but was inhibitory to cytochrome P450 loss due to vinyl chloride metabolism in microsomes from isoniazid-treated and untreated rats. These data demonstrate that the relatively metabolically inert solvent, 1,1,1-trichloroethane, can directly increase vinyl chloride metabolism and activation catalyzed by the phenobarbital-inducible isozymes, but is inhibitory toward vinyl chloride metabolism catalyzed by the isoniazid-inducible CYP2E1.

Extractive Derivatization of Aqueous Drugs Using Polymeric Phase Transfer Catalysts. I. Sensitive Analyses of Dialkyl Phosphates as Pentafluorobenzyl Derivatives.

The analyses of dialkylphosphates, aqueous metabolites or degradation products of organophosphorus pesticides, with extractive pentafluorobenzylation in the presence of polymeric phase-transfer catalysts (tri-phase catalysts) were investigated. Dialkylthiophosphates and dialkyldithiophosphates (alkyl=methyl, ethyl) in aqueous samples were extracted and derivatized in more than 98% yield by pentafluorobenzyl bromide in 15 min at 40°C using tri-n-butyl methyl phosphonium bromide polymer bound as a tri-phase catalyst and MIBK as an organic solvent. Dimethylphosphate and diethylphosphate were also derivatized in 43% and 100% yields, respectively, in the presence of this tri-phase catalyst in 90 min at 90°C. The simultaneous analyses of these 6 phosphates in the spiked urine sample were carried out with excellent detection limits, 0.44-0.79 ppm by scan-mode GC-MS and 0.027-0.081 ppm by ECD-GC) using this tri-phase procedure.

On the identification of cerebral metabolites in localized 1H NMR spectra of human brain In vivo

Localized 1H NMR spectra of human brain in vivo are affected by signal overlap, strong spin-spin coupling, and complex J modulation, and therefore differ considerably from those obtained at higher magnetic fields. This paper deals with the assignment of 1H NMR resonances of cerebral metabolites under the experimental conditions used for human investigations. Conventional 7.0-T FID spectra and 2.0 T localized, short echo time STEAM spectra (TE = 20 ms) of aqueous metabolite solutions are compared to in vivo brain spectra of human volunteers and patients. In addition to singlet resonances from N-acetyl aspartate (NAA), creatines, and cholines, short echo time STEAM spectra exhibit multiplets due to the NAA aspartyl group, glutamate, taurine, and myo-inositol. Enhanced levels of cerebral glutamine are detected in patients with liver cirrhosis. For the first time elevated levels of brain glucose are observed in patients with diabetes mellitus.