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Objective Stroke is a major cause of death and disability. That three-quarters of stroke patients will never have previously manifested cerebrovascular symptoms demonstrates the unmet clinical need for new biomarkers able to stratify patient risk and elucidation of the biological dysregulations. In this study, the utility of comprehensive metabolic phenotyping is assessed to provide candidate biomarkers that relate to stroke risk in stenosing carotid plaque tissue samples. Method Carotid plaque tissue samples were obtained from patients with cerebrovascular symptoms of carotid origin ( n = 5), and from asymptomatic patients ( n = 5). Two adjacent biological replicates were obtained from each tissue. Organic and aqueous metabolite extracts were obtained separately and analysed using two ultra performance liquid chromatography coupled to mass spectrometry metabolic profiling methods. Multivariate and univariate tools were used for statistical analysis. Results The two study groups demonstrated distinct plaque phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the two groups with a strong statistical significance ( p = 10 −4 –10 −5 ). Specifically, metabolites related to the eicosanoid pathway (arachidonic acid and arachidonic acid precursors), and three acylcarnitine species (butyrylcarnitine, hexanoylcarnitine, and palmitoylcarnitine), intermediates of the β-oxidation, were detected in higher intensities in symptomatic patients. However, metabolites implicated in the process of cell death, a process known to be upregulated in the formation of the vulnerable plaque, were unaffected. Conclusions Discrimination between symptomatic and asymptomatic carotid plaque tissue is demonstrated for the first time using metabolic profiling technologies. Two biological pathways (eicosanoid and β-oxidation) were implicated in differentiating symptomatic from asymptomatic patients and will be further investigated. These results indicate that metabolic phenotyping should be further explored to investigate the chemistry of the unstable plaque, in the pursuit of candidate biomarkers for risk-stratification and targets for pharmacotherapeutic intervention. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Nuclear magnetic resonance (NMR) spectroscopy is an established tool for metabolic profiling of tissues or biofluids with utility in identifying disease biomarkers and changes in enzymatic or gene expression. This pilot study aims to compare the metabolic profiles of intact varicose and non-varicose vein tissue via magic angle spinning (MAS) NMR spectroscopy with a view to promoting the understanding of the pathogenesis of varicose vein formation. Methods: Varicose vein tissue (n = 8) was collected from patients undergoing varicose veins surgery. Control non-varicose great saphenous vein samples were collected from patients undergoing lower limb amputation (n = 3) and peripheral arterial bypass surgery (n = 5). Intact tissue samples (average weight 10.33 ± 0.8 mg) from each vein segment were analysed using 1D MAS 1H NMR (600 MHz) spectroscopy. For selected vein samples, two-dimensional (2D) NMR experiments were performed. Differences between spectra from varicose and non-varicose tissues were elucidated using a variety of multivariate statistical analyses. Results: The metabolic profiles of varicose veins samples were clearly differentiated from non-varicose veins samples. Lipid metabolites were present at a higher concentration in the non-varicose veins group whilst creatine, lactate and myo-inositol metabolites were more characteristic of the varicose veins group. Conclusion: We demonstrate differential metabolic profiles between varicose veins and non-varicose veins. Elucidating the metabolic signature underlying varicose veins can further improve our understanding of the biological mechanisms of disease initiation, progression, and aid in identifying putative therapeutic targets. [Copyright &y& Elsevier]

Following the administration of 2-, 3- and 4-fluorobenzyl alcohols, the major metabolites detected in urine corresponded to the glycine conjugates of the corresponding benzoic acids. Little, or no, unchanged parent compound was detected in the samples. In addition to glycine-conjugated benzoic acids, a small proportion of the urinary metabolites for each of the fluorobenzyl alcohols was found to correspond to N-acetylcysteinyl conjugate. These were probably formed as the result of the production of a reactive sulphate ester during metabolism. The overall urinary recoveries of metabolites for the 2- and 3-fluorobenzyl alcohols were lower than that observed for the corresponding benzoic acids whilst that for 4-fluorobenzyl alcohol was similar. [Copyright &y& Elsevier]

High resolution magic angle spinning (HRMAS) 1H NMR spectroscopy was used to metabolically characterise Ishikawa cells, a human cell line derived from endometrial adenocarcinoma. The spectra obtained had well-resolved resonances from the nucleotide derivatives of uridine and adenosine. Using a combination of diffusion- and relaxation-weighted spectroscopy, the cellular environment of key metabolites previously identified as related to cell growth was also investigated. As Ishikawa cells are hormone-responsive, the metabolic action of tamoxifen, a selective estrogen receptor modulator (SERM), was also investigated. Cells were exposed to 5, 1 and 0.1 μM tamoxifen. Using the statistical regression technique of prediction to latent structures by partial least squares, a predictive model was built modelling the metabolic profile of the cells against exposure to tamoxifen. These spectral changes were characterised by increased resonance intensities from ethanolamine (3.26 ppm), glucose (3.34–3.94 ppm), glutamate (2.14, 2.32 ppm), tyrosine (7.24 ppm), uridine (7.85 ppm) and adenosine (8.20 ppm), and a relative decrease in contributions from myo-inositol resonances (3.30, 3.62, 3.55 ppm). The nucleotide changes suggest that tamoxifen affects RNA transcription, while the changes in ethanolamine and myo-inositol concentrations are indicative of cell membrane turnover. [Copyright &y& Elsevier]

1 H NMR Spectroscopy has been applied to determine the neurochemical profiles of brain extracts from the frontal cortex and hippocampal regions of germ free and normal mice and rats. The results revealed a number of differences between germ free (GF) and conventional (CV) rats or specific pathogen-free (SPF) mice with microbiome-associated metabolic variation found to be both species- and region-dependent. In the mouse, the GF frontal cortex contained lower amounts of creatine, N-acetyl-aspartate (NAA), glycerophosphocholine and lactate, but greater amounts of choline compared to that of specific pathogen free (SPF) mice. In the hippocampus, the GF mice had greater creatine, NAA, lactate and taurine content compared to those of the SPF animals, but lower relative quantities of succinate and an unidentified lipid-related component. The GF rat frontal cortex contained higher relative quantities of lactate, creatine and NAA compared to the CV animals whilst the GF hippocampus was characterized by higher taurine and phosphocholine concentrations and lower quantities of NAA, N-acetylaspartylglutamate and choline compared to the CV animals. Of note is that, in both rat and mouse brain extracts, concentrations of hippocampal taurine were found to be greater in the absence of an established microbiome. The results provide further evidence that brain biochemistry can be influenced by gut microbial status, specifically metabolites involved in energy metabolism demonstrating biochemical dialogue between the microbiome and brain. [ABSTRACT FROM AUTHOR]

Abstract: As end products of xenobiotic metabolism via glutathione conjugation, mercapturic acids (MCAs) can be used as markers to indicate exposure to allylic compounds as well as the rate and efficiency of their excretion. In addition, the formation of certain MCAs indicates metabolism via the known toxin acrolein, a strong electrophile. High-resolution 1H NMR spectroscopy has been employed to quantitatively measure the presence of MCAs in the urine of Sprague–Dawley rats, collected in the 8h following 25 and 50mgkg−1 i.p. doses of allyl formate (AF), a model toxin. 3-Hydroxypropylmercapturic acid (HPMA) was found to be the only 1H NMR-observable MCA excreted in the urine, exhibiting a percentage recovery of ∼20% at the 25mgkg−1 dose level, and ∼30% at the 50mgkg−1 dose level. [Copyright &y& Elsevier]

The urinary excretion profile and identity of the metabolites of 2-trifluoromethyl aniline (2-TFMA) and 2-trifluoromethyl acetanilide (2-TFMAc), following i.p. administration to the rat at 50 mg kg−1, were determined using a combination of 19F NMR monitored enzyme hydrolysis, SPEC-MS and 19F/1H HPLC-NMR. A total recovery of approximately 96.4% of the dose was excreted into the urine as seven metabolites. The major routes of metabolism were N-conjugation (glucuronidation), and ring-hydroxylation followed by sulphation (and to a lesser extent glucuronidation). The major metabolites excreted into the urine for both compounds were a labile N-conjugated metabolite (a postulated N-glucuronide) and a sulphated ring-hydroxylated metabolite (a postulated 4-amino-5-trifluoromethylphenyl sulphate) following dosing of 2-TFMA. These accounted for approximately 53.0 and 31.5% of the dose, respectively. This study identifies problems on sample component instability in the preparation and analysis procedures. [Copyright &y& Elsevier]

A combination of 19F, 1H NMR and HPLC-NMR spectroscopic approaches have been used to quantify and identify the urinary-excreted metabolites of 4-trifluoromethoxyaniline (4-TFMeA) and its [13C]-labelled acetanilide following i.p. administration at 50 mg/kg to rats. The major metabolite excreted in the urine for both compounds was a sulphated ring-hydroxylated metabolite (either 2- or 3-trifluoromethyl-5-aminosulphate) which accounted for ≈32.3% of the dose following the administration of 4-TFMeA and ≈29.9% following dosing of the acetanilide. The trifluoromethoxy-substituent appeared to be metabolically stable, with no evidence of O-detrifluoromethylation. There was no evidence of the excretion of N-oxanilic acids in urine, of the type seen with 4-trifluoromethylaniline. [Copyright &y& Elsevier]