Mouse Liver Extracts Research Articles

Direct Conversion of L-Selenomethionine into Methylselenol by Human Cystathionine ��-Lyase

Selenium is an essential trace element for mammals, but it is very toxic. Therefore, the control of selenium concentrations should be precisely and effectively monitored. Selenium is naturally obtained through foods and seleno-L-methionine (LSeMet) is a major form of selenium. It has been reported that L-SeMet is only converted into Se-adenosyl-L-SeMet. However, a recent study suggested that L-SeMet was directly metabolized into methylselenol () in mouse liver extract by the reaction of cystathionine -lyase (CGL). The canonical reaction of CGL was known to catalyze the cleavage of L-cystathionine to L-cysteine, -ketobutyrate and . In the present study, we found that L-SeMet could be directly converted to using purified homogenous human CGL instead of mouse liver cytosol. Authentic was prepared by reduction of dimethyldiselenide with sodium tetrahydroborate. The gaseous product of the enzymatic reaction with L-SeMet was analyzed by GC/MS spectrometry. The GC/MS data was identical to that of authentic dinitrophenyl selenoether. We also analyzed the kinetic parameters for the formation of from L-SeMet by human and mouse CGL. These results suggest that human CGL is a critical enzyme which is responsible for L-SeMet metabolism.

Power of isotopic fine structure for unambiguous determination of metabolite elemental compositions: In silico evaluation and metabolomic application

In mass spectrometry (MS)-based metabolomics studies, reference-free identification of metabolites is still a challenging issue. Previously, we demonstrated that the elemental composition (EC) of metabolites could be unambiguously determined using isotopic fine structure, observed by ultrahigh resolution MS, which provided the relative isotopic abundance (RIA) of 13C, 15N, 18O, and 34S. Herein, we evaluated the efficacy of the RIA for determining ECs based on the MS peaks of 20,258 known metabolites. The metabolites were simulated with a ≤25% error in the isotopic peak area to investigate how the error size effect affected the rate of unambiguous determination of the ECs. The simulation indicated that, in combination with reported constraint rules, the RIA led to unambiguous determination of the ECs for more than 90% of the tested metabolites. It was noteworthy that, in positive ion mode, the process could distinguish alkali metal-adduct ions ([M+Na]+ and [M+K]+). However, a significant degradation of the EC determination performance was observed when the method was applied to real metabolomic data (mouse liver extracts analyzed by infusion ESI), because of the influence of noise and bias on the RIA. To achieve ideal performance, as indicated in the simulation, we developed an additional method to compensate for bias on the measured ion intensities. The method improved the performance of the calculation, permitting determination of ECs for 72% of the observed peaks. The proposed method is considered a useful starting point for high-throughput identification of metabolites in metabolomic research.

Endogenous and xenobiotic metabolite profiling of liver extracts from SCID and chimeric humanized mice following repeated oral administration of troglitazone

1. Metabonomic analysis, via a combination of untargeted and targeted liquid chromatography-mass spectrometry (LC-MS) and untargeted 1H NMR spectroscopy-based metabolite profiling, was performed on aqueous (AQ) and organic liver extracts from control (SCID) and chimeric humanized (PXB) mice dosed with troglitazone at 0, 300 and 600 mg/kg/day for seven days.2. LC-MS analysis of AQ liver extracts showed a more “human-like” profile for troglitazone metabolites for PXB, compared with SCID, mice.3. LC-MS detected differences in endogenous metabolites, particularly lipid species in dosed mice, including elevated triacylglycerols and 1-alkyl,2-acylglycerophosphates as well as lowered diacylglycerophosphocholines and 1-alkyl,2-acylglycerophosphocholines for PXB compared with SCID mouse liver extracts. Following drug administration changes in the relative proportions of the ions for various unsaturated fatty acids were observed for both types of mouse, some of which were specific to PXB or SCID mice.4. 1H NMR spectroscopy revealed that AQ PXB mouse liver extracts had elevated amounts of inosine, fumarate, creatine, aspartate, trimethylamine N-oxide, glycerophosphocholine, phosphocholine, choline, glutamine, glutamate, acetate, alanine and lactate relative to SCID mice and decreased histidine, glycogen, α- and β-glucose, taurine, and glutathione. Increased uracil and tyrosine concentrations were detected for PXB mice on troglitazone administration.5. Metabonomic profiling thus showed clear differences between humanized and SCID mice, including after administration of troglitazone.

Penitrem A and analogues: toxicokinetics, toxicodynamics including mechanism of action and clinical significance

Penitrem A is a mycotoxin mainly produced by Penicillium crustosum, a fungal species occurring in all climate zones, ranging from tropical to arctic areas. P. crustosum produces a wide range of toxic metabolites, including penitrems, thomitrems and roquefortine C. The major metabolite, penitrem A, has been associated with several episodes of mycotoxicosis in dogs. The clinical symptoms of acute penitrem A intoxication include classical signs of neurotoxicity, such as tremors, convulsions, ataxia and nystagmus. The outcomes of penitrem A intoxication in animals range from total recovery to death, depending mainly on the level of exposure. Cases of suspected human mycotoxicosis following exposure to P. crustosum infected food, beer or inhalation of dust have also been reported. The toxicokinetics of penitrem A is scarcely studied. The toxin is rapidly absorbed, as demonstrated by the rapid onset of symptoms after exposure, but the absorption has not been quantified. Penitrem A is transported systemically after absorption and has been found in liver, kidney and brain as well as in serum and the gastrointestinal tract in exposed animals. Five phase I metabolites have been found in liver extracts of mice 60 min after oral exposure to penitrem A, while three metabolites were found after in vitro incubations with primary rat hepatocytes and rat liver microsomes. Only penitrem A was found in the brains of exposed mice or intoxicated dogs. The elimination has not been studied. Penitrem A is probably the main tremorgenic compound in Penicillium-infected food and feed commodities, since analogues had lower toxic potentials in comparative studies. Penitrem A affects the central as well as the peripheral nervous system. The toxin blocks the high-conductance Ca2+-activated potassium channels (BK) and impairs the GABAergic neurotransmission in the cerebellum. Animal poisoning by penitrem A is probably underdiagnosed due to a lack of knowledge among veterinarians.

Identification and Quantification of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Cells by Liquid Chromatography/Isotope-Dilution Tandem Mass Spectrometry

Unless repaired, DNA damage can drive mutagenesis or cell death. DNA repair proteins may therefore be used as biomarkers in disease etiology or therapeutic response prediction. Thus, the accurate determination of DNA repair protein expression and genotype is of fundamental importance. Among DNA repair proteins involved in base excision repair, apurinic/apyrimidinic endonuclease 1 (APE1) is the major endonuclease in mammals and plays important roles in transcriptional regulation and modulating stress responses. Here, we present a novel approach involving LC-MS/MS with isotope-dilution to positively identify and accurately quantify APE1 in human cells and mouse tissue. A completely 15N-labeled full-length human APE1 was produced and used as an internal standard. Fourteen tryptic peptides of both human APE1 (hAPE1) and 15N-labeled hAPE1 were identified following trypsin digestion. These peptides matched the theoretical peptides expected from trypsin digestion and provided a statistically significant protein score that would unequivocally identify hAPE1. Using the developed methodology, APE1 was positively identified and quantified in nuclear and cytoplasmic extracts of multiple human cell lines and mouse liver using selected-reaction monitoring of typical mass transitions of the tryptic peptides. We also show that the methodology can be applied to the identification of hAPE1 variants found in the human population. The results describe a novel approach for the accurate measurement of wild-type and variant forms of hAPE1 in vivo, and ultimately for defining the role of this protein in disease development and treatment responses.

Efficient chromatographic separation of intact proteins derivatized with a fluorogenic reagent for proteomics analysis

To achieve more efficient separation of intact proteins for proteomics applications, three columns of differing diameters (4.0, 4.6 and 6.0 mm internal diameter) were chosen for comparison and investigated to identify optimal conditions. The column with the largest diameter gave the largest peak capacity, showing the efficient separation of intact proteins, such as two protein standards, glutathione S-transferase and β-lactoglobulin. On the other hand, a low-molecular-weight compound was separated effectively on the smaller diameter column, demonstrating that the separation mechanism seems to differ between high- and low-molecular-weight compounds. Finally, using the 6.0 mm i.d. column, 680 protein peaks were observed in mouse liver extracts, demonstrating that a wider diameter separation column is effective for intact protein separations.

Identification of prolyl oligopeptidase as a cyclosporine-sensitive protease by screening of mouse liver extracts

Cyclosporine A (CsA) is a cyclic undecapeptide well known for its ability to prevent rejection episodes after organ transplantation via gain-of-function. Therefore, biomedical studies on CsA have been focused on both immunosuppressive properties and binding to the biocatalytically-active immune receptors, the cyclophilins. Much less attention has been spent on effects of cyclosporines on the biological function of other proteins. We used a 9-mer fluorescence-quenched peptide library with defined sequences to identify cyclosporine-sensitive proteolysis in mouse liver extracts. A highly soluble [d-Ser]8-CsA derivative was utilized to avoid drug precipitation at extended incubation times. Analysis of the time courses of proteolysis revealed 15 out of 360 peptide sequences where proteolysis exhibited marked sensitivity to the cyclosporine derivative. As a first example, a collagen-derived substrate was selected from those hits to identify the targeted proteolytic pathway. After purification from mouse liver extracts, prolyl oligopeptidase (EC 3.4.21.26) could be identified as a protease sensitive to submicromolar concentrations of cyclosporines. Surprisingly, in a series of cyclosporine derivatives an inverse relationship was found between the inhibition of prolyl oligopeptidase and inhibition of cyclophilin A.

In-depth Proteomic Characterization of Endogenous Nuclear Receptors in Mouse Liver

Nuclear receptors (NRs) are a superfamily of transcription factors that, upon binding to ligands, bind specific DNA sequences and regulate a transcriptional program governing cell proliferation, differentiation, and metabolism. In the liver, by sensing lipid-soluble hormones and dietary lipids and governing the expression of key liver metabolic genes, NR proteins direct a large array of key hepatic functions that include lipid and glucose metabolism, bile secretion, and bile acid homeostasis. Although much has been learned about the physiology of NRs, little is known about their protein expression and DNA binding activity in the liver because of their low abundance and the lack of high-throughput methods for detection at the protein level. Here we report a method for profiling the DNA binding activity of the NR transcription factor superfamily in mouse liver. We use DNA constructs of hormone response elements (HREs) as affinity reagents to enrich NR proteins from nuclear extracts of mouse liver and then identify them using mass spectrometry. We evaluated 20 DNA constructs containing various combinations of HREs for their ability to enrich endogenous NR proteins and found that two different HREs are sufficient to achieve isolation and identification of nearly all endogenous NR proteins from one mouse liver. We have detected proteins for 35 members of the NR family out of 41 that are expressed in mouse liver at mRNA level. Thus, this method allows coverage of most of the whole NR proteome and establishes a practical assay for the investigation of NR actions in mouse liver. We anticipate that this method will find widespread use in future investigations of NR actions in liver biology and pathology. Furthermore, this workflow is a useful tool for NR biologists interested in measuring NR expression, DNA binding, post-translational modifications, cellular localization, and other functional aspects of NRs in organs under normal physiological and pathological conditions, as well as during pharmacological intervention.

On-line two dimensional liquid chromatography/mass spectrometry for the analysis of triacylglycerides in peanut oil and mouse tissue

Triacylglycerides (TAGs) are a large class of complex neutral lipids that naturally occur in both plants and animals. In the present work, an on-line comprehensive silver-ion liquid chromatography (silver-ion LC)×reversed-phase liquid chromatography (RPLC) system was constructed to analyze these compounds. A micro bore silver-ion modified column was employed in the first dimension with the commonly used hexane-based mobile phase. After a series of C18 columns were assessed, a wide bore column packed with 1.5μm particles was selected as the second dimension column to reduce the negative effect caused by the large volume and strong solvent injection in the second dimension. The system coupled with mass spectrometry was applied to the analysis of an edible peanut oil and a mouse liver extract. Twenty-eight TAGs from the peanut oil and forty-four from the mouse liver were identified based on the TAGs’ retention behaviors on the comprehensive two-dimensional LC system and their APCI MS fragments.

Is the Kidney a Major Storage Site for Thyroxine as Thyroxine Glucuronide?

Previous studies have shown that thyroxine (T4) is stored as T4 glucuronide (T4G) in the kidney, and that 24 hours after administration of [(125)I]T4 to mice, 17% of the radioactivity was present in the kidneys, whereas only 4% was found in the liver. The present study was carried out to determine the relative amounts of conjugated and unconjugated T4 and 3,5,3'-triiodothyronine (T3) in the kidney and liver, and whether the conjugated hormones are extracted from tissues using our established extraction protocols, and detected in our radioimmunoassays (RIAs) for T4 and T3. Mice were injected with 10 μCi [(125)I]T4 or [(125)I]T3 and 24 hours later, the labeled compounds present in serum, kidney, liver, and urine were extracted and analyzed by paper chromatography before and after treatment with β-glucuronidase. In addition, the amounts of endogenous T4 and T3 in extracts of mouse kidney and liver were measured by RIA before and after treatment with β-glucuronidase. After [(125)I]T4, more than 95% of the total kidney and liver radioactivity was extracted, and in the kidney, almost all of it was present in a conjugated form, mostly as T4G. The liver also contained T4G, but none was present in serum or urine. T3 glucuronide (T3G) was also found in the kidney and liver after the administration of [(125)I]T3. Analysis by RIA of the endogenous T4 content in extracts of kidney before and after hydrolysis by β-glucuronidase revealed that a substantial fraction of the T4 in both tissues was present as T4G, and the T4G was not detected in the RIA. Furthermore, the combined T4+T4G content in the kidney expressed per gram of tissue was significantly higher than that in the liver or serum. In contrast, the kidney content of T3+T3G was very low compared with that of T4+T4G. In summary, we have shown that the kidney stores a significant amount of T4 as T4G. Since T4G deconjugation can occur rapidly in the kidney, it is possible that this tissue participates in maintaining extrathyroidal serum T4 homeostasis.

Ethylmalonyl-CoA Decarboxylase, a New Enzyme Involved in Metabolite Proofreading

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few such "metabolite proofreading enzymes" are known, our purpose was to search for an enzyme able to degrade ethylmalonyl-CoA, a potentially toxic metabolite formed at a low rate from butyryl-CoA by acetyl-CoA carboxylase and propionyl-CoA carboxylase, two major enzymes of lipid metabolism. We show that mammalian tissues contain a previously unknown enzyme that decarboxylates ethylmalonyl-CoA and, at lower rates, methylmalonyl-CoA but that does not act on malonyl-CoA. Ethylmalonyl-CoA decarboxylase is particularly abundant in brown adipose tissue, liver, and kidney in mice, and is essentially cytosolic. Because Escherichia coli methylmalonyl-CoA decarboxylase belongs to the family of enoyl-CoA hydratase (ECH), we searched mammalian databases for proteins of uncharacterized function belonging to the ECH family. Combining this database search approach with sequencing data obtained on a partially purified enzyme preparation, we identified ethylmalonyl-CoA decarboxylase as ECHDC1. We confirmed this identification by showing that recombinant mouse ECHDC1 has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity but no malonyl-CoA decarboxylase or enoyl-CoA hydratase activity. Furthermore, ECHDC1-specific siRNAs decreased the ethylmalonyl-CoA decarboxylase activity in human cells and increased the formation of ethylmalonate, most particularly in cells incubated with butyrate. These findings indicate that ethylmalonyl-CoA decarboxylase may correct a side activity of acetyl-CoA carboxylase and suggest that its mutation may be involved in the development of certain forms of ethylmalonic aciduria.

MetSign: a computational platform for high-resolution mass spectrometry-based metabolomics.

Data analysis in metabolomics is currently a major challenge, particularly when large sample sets are analyzed. Herein, we present a novel computational platform entitled MetSign for high-resolution mass spectrometry-based metabolomics. By converting the instrument raw data into mzXML format as its input data, MetSign provides a suite of bioinformatics tools to perform raw data deconvolution, metabolite putative assignment, peak list alignment, normalization, statistical significance tests, unsupervised pattern recognition, and time course analysis. MetSign uses a modular design and an interactive visual data mining approach to enable efficient extraction of useful patterns from data sets. Analysis steps, designed as containers, are presented with a wizard for the user to follow analyses. Each analysis step might contain multiple analysis procedures and/or methods and serves as a pausing point where users can interact with the system to review the results, to shape the next steps, and to return to previous steps to repeat them with different methods or parameter settings. Analysis of metabolite extract of mouse liver with spiked-in acid standards shows that MetSign outperforms the existing publically available software packages. MetSign has also been successfully applied to investigate the regulation and time course trajectory of metabolites in hepatic liver.

Abstract A63: TLR5 agonist CBLB502 protects mice from Fas-mediated liver damage without protecting tumors

Abstract We have previously shown the strong radioprotective properties of the Toll-like receptor 5 (TLR5) agonist flagellin and its derivative CBLB502 in both mouse and non-human primate models (Burdelya et al., 2008, Science 320: 226–230). Because no protection has been observed in any in vitro cell model regardless of the TLR5 status of the cells, we hypothesized that the radioprotective effects of the TLR5 agonist are indirect and mediated by factors produced by primary cells responding to flagellin in the organism. Using a combination of tools, including NF-kB-responsive reporter mice and immunohistochemical determination of tissues responding to CBLB502 by NF-kB activation, we defined liver hepatocytes as the primary pharmacological targets of flagellin. Surgical exclusion of the liver from the blood circulation completely abolished radioprotection of the bone marrow hematopoietic progenitor cells by CBLB502 suggesting that the liver acts as a mediator of at least some of the effects of the TLR5 agonist. Since the induction of NF-kB is known to activate multiple pro-survival mechanisms, we further hypothesized that administration of the TLR5 agonist can result in liver resistance to otherwise toxic conditions. Mice treated with Fas agonistic antibodies were employed as a model of acute hepatotoxicity. Injection of CBLB502 showed a strong preventive effect against Fas-mediated injury rescuing all of the injected mice in contrast to complete lethality in the control groups. The ability of CBLB502 to protect the liver was also demonstrated via reduced levels of liver enzymes in the peripheral blood and caspase activation in liver extracts of mice treated with Fas agonistic antibodies. Using two syngeneic tumor models, colon adenocarcinoma CT26 and lymphoma A20, growing as liver metastases in mice, we found that CBLB502 injections not only prevented liver damage, but also suppressed tumor appearance and growth when injected with or without anti-Fas antibodies. Thus, TLR5 agonist has potential to become a new agent for liver protection and suppression of liver metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A63.

Regulation of TFEB and V-ATPases by mTORC1

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is an important, highly conserved, regulator of cell growth. Ancient among the signals that regulate mTORC1 are nutrients. Amino acids direct mTORC1 to the surface of the late endosome/lysosome, where mTORC1 becomes receptive to other inputs. However, the interplay between endosomes and mTORC1 is poorly understood. Here, we report the discovery of a network that links mTORC1 to a critical component of the late endosome/lysosome, the V-ATPase. In an unbiased screen, we found that mTORC1 regulated the expression of, among other lysosomal genes, the V-ATPases. mTORC1 regulates V-ATPase expression both in cells and in mice. V-ATPase regulation by mTORC1 involves a transcription factor translocated in renal cancer, TFEB. TFEB is required for the expression of a large subset of mTORC1 responsive genes. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization and in a manner dependent on both TFEB and V-ATPases, mTORC1 promotes endocytosis. These data uncover a regulatory network linking an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis.

MicroRNA links obesity and impaired glucose metabolism

MicroRNA links obesity and impaired glucose metabolism

Erratum: CD95 promotes tumour growth

Nature 465, 492–496 (2010) In this Letter, an experimental error affected the western blot analyses of mouse liver extracts shown in Fig. 4f and g. The secondary antibody cross-reacted with endogenous mouse IgG in the tissue lysates, resulting in incorrect bands. The experiments were repeated using different primary antibodies and a secondary antibody that showed no such cross-reactivity.

Analysis of changes in triacylglycerol ratios in mouse liver and plasma in response to a liver X receptor agonist

An LC-MS method was developed for the analysis of triacylglycerols (TAG) in mouse liver extracts and plasma samples. C57 Mice were treated with two LXR agonists that have been shown to upregulate TAGs, T0901317 (T1317) or Org 264693 and compared to vehicle dosed animals. The dose used was 30 mg kg−1, once daily, with three different dose regimes; 24 H, 48 H and 5 day. The TAG ratios measured were C52:2/C54:3 and C52:3/C54:4, which corresponded to a decrease in the palmitate and an increase in oleate composition of the TAGs. A significant change in the C52:2/C54:3 ratio was observed with all dose regimes and a good correlation was obtained between liver and plasma samples. In a separate study, the same compounds were dosed to LXR α and LXR β knock-out (KO) mice at 30 mg kg−1, once daily, for 5 days. The LXR β KO mice showed similar TAG ratio changes to the C57 mice, whereas the LXR α KO mice showed no change in TAG ratios versus vehicle dosed animals. Measurements of lipid liability in response to an LXR agonist are typically made by measuring total liver TAG levels, which here, only showed a significant effect after the 48 H and 5 day dose regimes. By using a ratio measurement analysis could be performed on plasma samples, greatly simplifying the sample preparation procedure, without the requirement for either calibration curves or an internal standard.

Measuring DNA repair incision activity of mouse tissue extracts towards singlet oxygen-induced DNA damage: a comet-based in vitro repair assay

During the past two decades, the comet-based in vitro DNA repair assay has been used regularly to measure base excision repair (BER)-related DNA incision activity. Most studies focus on the assessment of BER in human lymphocytes or cultured cells by estimating the activity of a cell extract on substrate DNA containing specific lesions such as 8-oxoguanine. However, for many 'real-life' studies, it would be preferable to measure BER in the tissues of interest instead of using in vitro models or surrogate 'tissues' such as lymphocytes. Various attempts have been made to use the comet-based repair assay for BER with extracts from rodent tissues, but high non-specific nuclease activity in such tissues were a significant impediment to robust estimates of BER. Our aim in this study was to optimise the in vitro repair assay for BER for use with rodent tissues using extracts from liver and brain from C57/BL mice. Because the DNA incision activity of an extract is dependent on its protein concentration, the first optimisation step in preventing interference by non-specific nuclease activity was to determine the protein concentration at which there is a maximal difference between the total and non-specific damage recognition. This protein concentration was 5 mg/ml for mouse liver extracts and 1 mg/ml for brain extracts. Next, we tested addition of proteinase inhibitors during the preparation of the tissue extracts, but this did not improve the sensitivity of the assay. However, addition of 1.5 μM aphidicolin to the tissue extracts improved the detection of DNA repair incision activity by reducing non-specific nuclease activity and possibly by blocking residual DNA polymerase activity. Finally, the assay was tested on tissue samples from an ageing mouse colony and in mice undergoing dietary restriction and proved capable of detecting significant inter-animal differences and nutritional effects on BER-related DNA incision activity.

Mass spectrometry based metabolomics to study the development of Non-alcoholic steatohepatitis

It can be hypothesized that the development of non-alcoholic steatohepatitis (NASH) is accompanied by severe changes at the metabolic level. Studying the metabolome may reveal insights in the underlying mechanisms leading from steatosis to the development of NASH and its progression to cirrhosis. Metabolomics attempts the comprehensive and quantitative analysis of metabolites in a biological system. Two complementary approaches are used for metabolomic investigations: metabolic fingerprinting and metabolic profiling. Metabolic fingerprinting aims to identify patterns or “fingerprints“ of metabolites, which change in response to perturbations. In metabolic profiling, quantitative analytical methods are developed for metabolites in a metabolic pathway.This contribution will present two robust analytical methods for metabolic profiling and metabolic fingerprinting that were used to quantitatively probe changes in the metabolome associated with the development of NASH. Metabolic fingerprinting was performed by gas chromatography coupled to mass spectrometry (GC-MS). Furthermore, an LC–ESI-MS/MS method was developed for the quantitative determination of 14 intermediates of the methionine and polyamine pathways with lower limits of quantification in the range of 5.0–500 nM [1]. Preliminary studies were performed with samples from a long-term feeding study. BALB/c mice were fed a control or a high fat diet. Liver tissue extracts were subjected to metabolic fingerprinting by GC-MS. A clear segregation of the two groups was observed in a principal component analysis. The identification of discriminating metabolites is in progress. Metabolic changes in the methionine and polyamine pathways were analyzed in methanolic liver extracts. The preliminary data from the liver extracts of mice fed the paigen diet show a significant increase for purescine and 5-deoxy-5-(methylthio)adenosine, while S-adenosyl -homocysteine, spermidine, spermine and acetylspermine were down regulated.

In situ analysis of [8-13C-7-15N]-double-labelled theophylline by a triple resonance NMR technique

[8-13C-7-15N]-labelled theophylline was synthesized and applied for in situ analysis using a 1H–{13C–15N} triple resonance NMR technique. The triple resonance analysis enabled selective detection of labelled theophylline in a mixture containing excess amino acids or crude mouse liver extract, where conventional 1H NMR analysis suffers from difficulty because of background signals.