Exploring extra dimensions to capture saliva metabolite fingerprints from metabolically healthy and unhealthy obese patients by comprehensive two-dimensional gas chromatography featuring Tandem Ionization mass spectrometry.

This study examines the information potential of comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC×GC-TOF MS) and variable ionization energy (i.e., Tandem Ionization™) to study changes in saliva metabolic signatures from a small group of obese individuals. The study presents a proof of concept for an effective exploitation of the complementary nature of tandem ionization data. Samples are taken from two sub-populations of severely obese (BMI > 40 kg/m2) patients, named metabolically healthy obese (MHO) and metabolically unhealthy obese (MUO). Untargeted fingerprinting, based on pattern recognition by template matching, is applied on single data streams and on fused data, obtained by combining raw signals from the two ionization energies (12 and 70 eV). Results indicate that at lower energy (i.e., 12 eV), the total signal intensity is one order of magnitude lower compared to the reference signal at 70 eV, but the ranges of variations for 2D peak responses is larger, extending the dynamic range. Fused data combine benefits from 70 eV and 12 eV resulting in more comprehensive coverage by sample fingerprints. Multivariate statistics, principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) show quite good patient clustering, with total explained variance by the first two principal components (PCs) that increases from 54% at 70 eV to 59% at 12 eV and up to 71% for fused data. With PLS-DA, discriminant components are highlighted and putatively identified by comparing retention data and 70 eV spectral signatures. Within the most informative analytes, lactose is present in higher relative amount in saliva from MHO patients, whereas N-acetyl-D-glucosamine, urea, glucuronic acid γ-lactone, 2-deoxyribose, N-acetylneuraminic acid methyl ester, and 5-aminovaleric acid are more abundant in MUO patients. Visual feature fingerprinting is combined with pattern recognition algorithms to highlight metabolite variations between composite per-class images obtained by combining raw data from individuals belonging to different classes, i.e., MUO vs. MHO.Graphical abstract.

Whole grain intake associated molecule 5-aminovaleric acid betaine decreases ß-oxidation of fatty acids in mouse cardiomyocytes.

Despite epidemiological evidence showing that diets rich in whole grains reduce the risk of chronic life-style related diseases, biological mechanisms for these positive effects are mostly unknown. Increased 5-aminovaleric acid betaine (5-AVAB) levels in plasma and metabolically active tissues such as heart have been associated with consumption of diets rich in whole grains. However, biological effects of 5-AVAB are poorly understood. We evaluated 5-AVAB concentrations in human and mouse heart tissue (3-22 µM and 38-78 µM, respectively) using mass spectrometry. We show that 5-AVAB, at physiological concentration range, dose-dependently inhibits oxygen consumption due to ß-oxidation of fatty acids, but does not otherwise compromise mitochondrial respiration, as measured with oxygen consumption rate in cultured mouse primary cardiomyocytes. We also demonstrate that this effect is caused by 5-AVAB induced reduction of cellular L-carnitine. Reduced L-carnitine levels are at least partly mediated by the inhibition of cell membrane carnitine transporter (OCTN2) as evaluated by in silico docking, and by siRNA mediated silencing of OCTN2 in cultured cardiomyocytes. 5-AVAB caused inhibition of ß-oxidation of fatty acids is a novel mechanism on how diets rich in whole grains may regulate energy metabolism in the body. Elucidating potentially beneficial effects of 5-AVAB e.g. on cardiac physiology will require further in vivo investigations.

Thermo-alkaline Treatment as a Practical Degradation Strategy To Reduce Indospicine Contamination in Camel Meat.

Ingestion of indospicine-contaminated camel and horse meat has caused fatal liver injury to dogs in Australia, and it is currently not known if such contaminated meat may pose a human health risk upon dietary exposure. To date, indospicine-related research has tended to focus on analytical aspects, with little information on post-harvest management of indospicine-contaminated meat. In this study, indospicine degradation was investigated in both aqueous solution and also contaminated meat, under a range of conditions. Aqueous solutions of indospicine and indospicine-contaminated camel meat were microwaved (180 °C) or autoclaved (121 °C) with the addition of food-grade additives [0.05% (v/v) acetic acid or 0.05% (w/v) sodium bicarbonate] for 0, 15, 30, and 60 min. An aqueous sodium bicarbonate solution demonstrated the greatest efficacy in degrading indospicine, with complete degradation after 15 min of heating in a microwave or autoclave; concomitant formation of indospicine degradation products, namely, 2-aminopimelamic and 2-aminopimelic acids, was observed. Similar treatment of indospicine-contaminated camel meat with aqueous sodium bicarbonate resulted in 50% degradation after 15 min of heating in an autoclave and 100% degradation after 15 min of heating in a microwave. The results suggest that thermo-alkaline aqueous treatment has potential as a pragmatic post-harvest handling technique in reducing indospicine levels in indospicine-contaminated meat.

Protein substitutes for phenylketonuria in Europe: access and nutritional composition.

BACKGROUND/OBJECTIVES: Protein substitutes (PS) are an essential component in the dietary management of phenylketonuria (PKU). PS are available as phenylalanine-free amino-acid mixtures (AAM), glycomacropeptide-based PS (GMP) and large neutral amino acids (LNAA). There is a lack of information regarding their availability in different countries and comparison of their nutritional composition is limited. The objectives of this study were to identify the number of PS available in different European countries and Turkey and to compare their nutritional composition. SUBJECTS/METHODS: Members of the European Nutritionist Expert Panel on PKU (ENEP) (Portugal, Spain, Belgium, Italy, Germany, Netherlands, United Kingdom, Denmark and Turkey) provided data on PS available in each country. The nutritional composition of PS available in Portugal was analyzed. RESULTS: The number of PS available in each country varied from 30 (Turkey) to 105 (Germany), with a median of 64. GMP was available only in Portugal, whereas LNAA was an option in Portugal, Italy, Turkey and Denmark. Some PS were designed for weaning. Many PS did not contain added fat and fiber. GMP contained the highest carbohydrate (CHO) and energy content as well as higher LNAA content compared with AAM. Only one AAM contained added fructo-oligosaccharides and galacto-oligosaccharides. AAM designed for the first year of life had the highest CHO, fat and LNAA contribution. Liquid AAM had lower CHO and fat contents compared with powdered AAM, but contained higher LNAA. CONCLUSIONS: There was widely dissimilar numbers of PS available in different countries. Nutritional composition of different PS was variable and should be considered before prescription.

Analysis of Betaines from Marine Algae Using LC-MS-MS.

Betaines are a class of quaternary ammonium compounds found in marine algae that can act as osmolytes and/or affect gene expression, and therefore improve plant tolerance to stresses such as temperature extremes, drought, and salinity when applied to agricultural crops. In humans, glycine betaine acts as a methyl donor and has been shown to protect internal organs, improve vascular risk factors, and enhance sport performance. Here we describe a sensitive LC-MS-MS method for the baseline separation and quantification of four betaines found in algae, namely, glycine betaine, δ-aminovaleric acid betaine, γ-aminobutyric acid betaine, and laminine.

High-level conversion of L-lysine into 5-aminovalerate that can be used for nylon 6,5 synthesis.

L-Lysine is a potential feedstock for the production of bio-based precursors for engineering plastics. In this study, we developed a microbial process for high-level conversion of L-lysine into 5-aminovalerate (5AVA) that can be used as a monomer in nylon 6,5 synthesis. Recombinant Escherichia coli WL3110 strain expressing Pseudomonas putida delta-aminovaleramidase (DavA) and lysine 2-monooxygenase (DavB) was grown to high density in fed-batch culture and used as a whole cell catalyst. High-density E. coli WL3110 expressing DavAB, grown to an optical density at 600 nm (OD600 ) of 30, yielded 36.51 g/L 5AVA from 60 g/L L-lysine in 24 h. Doubling the cell density of E. coli WL3110 improved the conversion yield to 47.96 g/L 5AVA from 60 g/L of L-lysine in 24 h. 5AVA production was further improved by doubling the L-lysine concentration from 60 to 120 g/L. The highest 5AVA titer (90.59 g/L; molar yield 0.942) was obtained from 120 g/L L-lysine by E. coli WL3110 cells grown to OD600 of 60. Finally, nylon 6,5 was synthesized by bulk polymerization of ϵ-caprolactam and δ-valerolactam prepared from microbially synthesized 5AVA. The hybrid system demonstrated here has promising possibilities for application in the development of industrial bio-nylon production processes.

A surgical loupe system for observing protoporphyrin IX fluorescence in high-grade gliomas after administering 5-aminolevulinic acid.

BACKGROUND: We recently developed a surgical loupe system for observing the fluorescence emitted by protoporphyrin IX (PpIX), a metabolite of 5-aminolevulinic acid. METHODS: This system used a semiconductor laser as the excitation light source. A compact, transparent, and ultraviolet cut-off filter was mounted on an eyepiece lens, which did not require filter on-off manipulation. RESULTS: Good quality protoporphyrin IX fluorescence was acquired using the surgical loupe system during glioblastoma resection, which was nearly identical to that acquired by fluorescent microscopy. In addition, surgeons can perform ordinary surgical procedures using this surgical loupe system under white light. CONCLUSION: This surgical loupe system enables the detection of PpIX fluorescence during resection of high-grade glioma. Further evaluations of this system are required to determine the extent of surgical resection before its practical application.

Engineering Escherichia coli for renewable production of the 5-carbon polyamide building-blocks 5-aminovalerate and glutarate.

Through metabolic pathway engineering, novel microbial biocatalysts can be engineered to convert renewable resources into useful chemicals, including monomer building-blocks for bioplastics production. Here we describe the systematic engineering of Escherichia coli to produce, as individual products, two 5-carbon polyamide building blocks, namely 5-aminovalerate (AMV) and glutarate. The modular pathways were derived using "parts" from the natural lysine degradation pathway of Pseudomonas putida KT2440. Endogenous over-production of the required precursor, lysine, was first achieved through metabolic deregulation of its biosynthesis pathway by introducing feedback resistant mutants of aspartate kinase III and dihydrodipicolinate synthase. Further disruption of native lysine decarboxylase activity (by deleting cadA and ldcC) limited cadaverine by-product formation, enabling lysine production to 2.25 g/L at a glucose yield of 138 mmol/mol (18% of theoretical). Co-expression of lysine monooxygenase and 5-aminovaleramide amidohydrolase (encoded by davBA) then resulted in the production of 0.86 g/L AMV in 48 h. Finally, the additional co-expression of glutaric semialdehyde dehydrogenase and 5-aminovalerate aminotransferase (encoded by davDT) led to the production of 0.82 g/L glutarate under the same conditions. At this output, yields on glucose were 71 and 68 mmol/mol for AMV and glutarate (9.5 and 9.1% of theoretical), respectively. These findings further expand the number and diversity of polyamide monomers that can be derived directly from renewable resources.

Aminoácidos en líquido cefalorraquídeo y plasma: utilidad en el estudio de las enfermedades neuropediátricas / Amino acids in cerebrospinal fluid and plasma: its usefulness in the study of neuropaediatric diseases

Introducción. El estudio de aminoácidos en el líquido cefalorraquídeo (LCR) es imprescindible en el diagnóstico de algunas enfermedades neurológicas y apoya el diagnóstico en otras. No existen trabajos en la bibliografía que muestren la relación fisiológica entre los valores de aminoácidos en LCR y plasma en población pediátrica. Objetivo. Definir unas ratios de aminoácidos en plasma y LCR en población pediátrica que permitan su uso en la práctica clínica diaria. Pacientes y métodos. Se han recogido y analizado de forma retrospectiva los aminogramas en plasma y LCR de 105 pacientes de edades comprendidas entre 0 y 12 meses. Se han incluido aminogramas cuyos valores de aminoácidos son normales según los valores de referencia de nuestro laboratorio. El análisis cuantitativo de aminoácidos se realizó mediante cromatografía líquida de alta resolución, y el análisis estadístico, con el programa SPSS v. 19.0. Resultados. Se muestran los valores medios, rango y desviación estándar de las concentraciones de aminoácidos en plasma y LCR, así como de las ratios LCR/plasma. Se han encontrado correlaciones significativas a partir de 0,6 entre diferentes aminoácidos neutros, sobre todo los de peso molecular más pequeño (Thr, Ser, Gly y Ala). Conclusiones. La existencia de correlaciones significativas entre diferentes aminoácidos neutros apoya el hecho de que éstos compartan los mismos transportadores en la barrera hematoencefálica. La estandarización de ratios de aminoácidos permitirá aumentar la sensibilidad en la detección de valores patológicos en plasma y LCR, profundizar en la fisiopatología de enfermedades neurológicas y quizá describir nuevas aminoacidopatías (AU)

Introduction. Studying the amino acids in cerebrospinal fluid (CSF) is essential in the diagnosis of some neurological diseases and is an important aid in the diagnosis of others. No research has been published in the literature to prove the physiological relationship between the values of amino acids in CSF and plasma in the paediatric population. Aim. To define a set of ratios for amino acids in plasma and CSF in the paediatric population that can be used in daily clinical practice. Patients and methods. The aminograms in plasma and CSF of 105 patients with ages between 0 and 12 months were collected and analysed retrospectively. Aminograms with amino acid values that are considered to be normal according to the reference values of our laboratory were included in the sample. The quantitative analysis of amino acids was performed using high-resolution liquid chromatography and statistical analysis with the software application SPSS 19.0. Results. The mean values, range and standard deviation of the amino acid concentrations in plasma and CSF, together with the CSF/plasma ratios, are reported. Significant correlations were found from 0.6 onwards between different neutral amino acids, above all in those with smaller molecular weights (Thr, Ser, Gly and Ala). Conclusions. The existence of significant correlations between the different neutral amino acids supports the idea that they share the same transporters in the blood-brain barrier. Standardising the amino acid ratios will make it possible to increase sensitivity in the detection of pathological values in plasma and CSF, to further knowledge of the pathophysiology of neurological diseases and perhaps to describe new aminoacidopathies (AU)

Systemic multicompartmental effects of the gut microbiome on mouse metabolic phenotypes.

To characterize the impact of gut microbiota on host metabolism, we investigated the multicompartmental metabolic profiles of a conventional mouse strain (C3H/HeJ) (n=5) and its germ-free (GF) equivalent (n=5). We confirm that the microbiome strongly impacts on the metabolism of bile acids through the enterohepatic cycle and gut metabolism (higher levels of phosphocholine and glycine in GF liver and marked higher levels of bile acids in three gut compartments). Furthermore we demonstrate that (1) well-defined metabolic differences exist in all examined compartments between the metabotypes of GF and conventional mice: bacterial co-metabolic products such as hippurate (urine) and 5-aminovalerate (colon epithelium) were found at reduced concentrations, whereas raffinose was only detected in GF colonic profiles. (2) The microbiome also influences kidney homeostasis with elevated levels of key cell volume regulators (betaine, choline, myo-inositol and so on) observed in GF kidneys. (3) Gut microbiota modulate metabotype expression at both local (gut) and global (biofluids, kidney, liver) system levels and hence influence the responses to a variety of dietary modulation and drug exposures relevant to personalized health-care investigations.

Convenient method for the determination of arginine and its related compounds in rumen fluid by reversed-phase high-performance liquid chromatography.

In order to clarify arginine (Arg) metabolism by rumen microorganisms and by the tissues of ruminant animals, a convenient method for the simultaneous determination of Arg, citrulline (Cit), ornithine (Orn), proline (Pro) and 5-aminovaleric acid (5AV), and 4-aminobutyric acid (4AB) and lysine (Lys), incidentally, in goat rumen fluid was established by reversed-phase high-performance liquid chromatography (RP-HPLC). The separation was carried out by stepwise isocratic elution with two mobile phases (solvent A and solvent B) on a LiChrospher 100 RP-18 column (150x4.6 mm I.D., 5 microm particle size) equipped with a guard column (4.0x4 mm, 5 microm particle size). Solvent A is composed of acetonitrile-sodium citrate buffer (pH 7.2) (15:85, v/v) containing tetrahydrofuran (5 ml/100 ml), with solvent B comprising acetonitrile-sodium citrate buffer (pH 5.4) (40:60, v/v). Five compounds (Cit, Arg, Pro, 4AB and 5AV) were separated within 33 min in solvent A and the other two (Orn and Lys) in solvent B. Solvent A was automatically switched to solvent B with the help of a valve controller. Complete separation needs 62 min after sample injection in a single chromatogram. Samples were derivatized with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) and detected on a fluorescence detector at excitation and emission wavelengths of 263 and 611 nm, respectively. The minimum detectable concentrations (microM) (signal-to-noise ratio, S/N 3:1) of these compounds were: 0.65 for Cit, 0.65 for Arg, 1.9 for Pro, 1.3 for 4AB, 1.9 for 5AV, 0.12 for Orn and 0.48 for Lys. When applied to rumen fluid from goats, recoveries of all compounds added to the rumen fluid were 96.6-100.6% for an intra-day study and 93.9-99.4% for inter-day (5 days) studies. The average contents of Orn, 5AV and Lys in the rumen fluid of three goats before morning feeding were 7.3, 13.5 and 3.6 microM, but Cit, Arg, Pro, and 4AB were not found, although all these four compounds were detected 1 h after feeding. Pro (390 microM) and 5AV (497.6 microM) were highest 1 h after feeding and then decreased. Orn levels before morning feeding were most similar to those after feeding.

Distribution volume of 3-O-methyl-6.

The distribution volume (DV) of 6-[F-18]fluoro-L-DOPA (FDOPA) in the cerebellum recently has been linked using positron emission tomography (PET) to plasma large neutral amino acid (LNAA) concentrations in monkeys. In this article the authors provide additional experimental support for this relation by directly measuring the DV as the steady-state tissue to plasma radioactivity ratio in rats using a labeled LNAA analog 3-O-methyl-6-[F-18]FDOPA (OMFD), a compound that has no known specific enzyme or receptor interactions in brain tissue. The measured DV for OMFD (tissue OMFD concentration/plasma OMFD concentration) was found to be inversely related to plasma LNAA concentrations. The relation (DV = 1.5-0.00094*[LNAA], R--2 = 0.79) resulted in an 8% DV decrease per 100 nmol/mL plasma LNAA increase within the observed range of 330 to 510 nmol/mL. This was similar to recent noninvasive observations with FDOPA PET in vervet monkeys and with 6-[F-18]Fluoro-m-tyrosine PET in squirrel monkeys. The OMFD striatum to cerebellum (Str/Cb) ratio was greater than 1.0 for all measurements, averaging 1.09 +/- 0.04, and was approximately equal to the Str/Cb LNAA ratio of 1.12 +/- 0.05. This current study verifies the variation of DV of OMFD or FDOPA as a function of plasma LNAA concentrations and suggests the possibility of using OMFD for measuring cerebral LNAA noninvasively with PET.