Chronic exercise improves hepatic acylcarnitine handling.

Exercise mediates tissue metabolic function through direct and indirect adaptations to acylcarnitine (AC) metabolism, but the exact mechanisms are unclear. We found that circulating medium-chain acylcarnitines (AC) (C12-C16) are lower in active/endurance trained human subjects compared to sedentary controls, and this is correlated with elevated cardiorespiratory fitness and reduced adiposity. In mice, exercise reduced serum AC and increased liver AC, and this was accompanied by a marked increase in expression of genes involved in hepatic AC metabolism and mitochondrial ß-oxidation. Primary hepatocytes from high-fat fed, exercise trained mice had increased basal respiration compared to hepatocytes from high-fat fed sedentary mice, which may be attributed to increased Ca2+ cycling and lipid uptake into mitochondria. The addition of specific medium- and long-chain AC to sedentary hepatocytes increased mitochondrial respiration, mirroring the exercise phenotype. These data indicate that AC redistribution is an exercise-induced mechanism to improve hepatic function and metabolism.

Differential oxidative metabolism and 5-ketoclomazone accumulation are involved in Echinochloa phyllopogon resistance to clomazone.

Echinochloa phyllopogon (late watergrass) is a major weed of California rice (Oryza sativa) that has evolved cytochrome P450-mediated metabolic resistance to different herbicides with multiple modes of action. E. phyllopogon populations from Sacramento Valley rice fields have also recently shown resistance to the herbicide clomazone. Clomazone is a proherbicide that must be metabolized to 5-ketoclomazone, which is the active compound that inhibits deoxyxylulose 5-phosphate synthase, a key enzyme of the nonmevalonate isoprenoid pathway. This study evaluated the differential clomazone metabolism within strains of the same species to investigate whether enhanced oxidative metabolism also confers clomazone resistance in E. phyllopogon. Using reverse-phase liquid chromatography-tandem mass spectrometry techniques in the multireaction monitoring mode, we elucidated that oxidative biotransformations are involved as a mechanism of clomazone resistance in this species. E. phyllopogon plants hydroxylated mostly the isoxazolidinone ring of clomazone, and clomazone hydroxylation activity was greater in resistant than in susceptible plants. The major clomazone metabolites resulted from monohydroxylation and dihydroxylation of the isoxazolidinone ring. Resistant plants accumulated 6- to 12-fold more of the monohydroxylated metabolite than susceptible plants, while susceptible plants accumulated 2.5-fold more of the phytotoxic metabolite of clomazone, 5-ketoclomazone. Our results demonstrate that oxidative metabolism endows multiple-herbicide-resistant E. phyllopogon with cross-resistance to clomazone through enhanced herbicide degradation and lower accumulation of the toxic metabolite in resistant versus susceptible plants.

Older adults with sarcopenia have distinct skeletal muscle phosphodiester, phosphocreatine, and phospholipid profiles.

The loss of skeletal muscle mass and function with age (sarcopenia) is a critical healthcare challenge for older adults. 31-phosphorus magnetic resonance spectroscopy (31 P-MRS) is a powerful tool used to evaluate phosphorus metabolite levels in muscle. Here, we sought to determine which phosphorus metabolites were linked with reduced muscle mass and function in older adults. This investigation was conducted across two separate studies. Resting phosphorus metabolites in skeletal muscle were examined by 31 P-MRS. In the first study, fifty-five older adults with obesity were enrolled and we found that resting phosphocreatine (PCr) was positively associated with muscle volume and knee extensor peak power, while a phosphodiester peak (PDE2) was negatively related to these variables. In the second study, we examined well-phenotyped older adults that were classified as nonsarcopenic or sarcopenic based on sex-specific criteria described by the European Working Group on Sarcopenia in Older People. PCr content was lower in muscle from older adults with sarcopenia compared to controls, while PDE2 was elevated. Percutaneous biopsy specimens of the vastus lateralis were obtained for metabolomic and lipidomic analyses. Lower PCr was related to higher muscle creatine. PDE2 was associated with glycerol-phosphoethanolamine levels, a putative marker of phospholipid membrane damage. Lipidomic analyses revealed that the major phospholipids, (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol) were elevated in sarcopenic muscle and were inversely related to muscle volume and peak power. These data suggest phosphorus metabolites and phospholipids are associated with the loss of skeletal muscle mass and function in older adults.

Skeletal Muscle Energetics and Mitochondrial Function Are Impaired Following 10 Days of Bed Rest in Older Adults.

BACKGROUND: Older adults exposed to periods of inactivity during hospitalization, illness, or injury lose muscle mass and strength. This, in turn, predisposes poor recovery of physical function upon reambulation and represents a significant health risk for older adults. Bed rest (BR) results in altered skeletal muscle fuel metabolism and loss of oxidative capacity that have recently been linked to the muscle atrophy program. Our primary objective was to explore the effects of BR on mitochondrial energetics in muscle from older adults. A secondary objective was to examine the effect of ß-hydroxy-ß-methylbuturate (HMB) supplementation on mitochondrial energetics. METHODS: We studied 20 older adults before and after a 10-day BR intervention, who consumed a complete oral nutritional supplement (ONS) with HMB (3.0 g/d HMB, n = 11) or without HMB (CON, n = 9). Percutaneous biopsies of the vastus lateralis were obtained to determine mitochondrial respiration and H2O2 emission in permeabilized muscle fibers along with markers of content. RNA sequencing and lipidomics analyses were also conducted. RESULTS: We found a significant up-regulation of collagen synthesis and down-regulation of ribosome, oxidative metabolism and mitochondrial gene transcripts following BR in the CON group. Alterations to these gene transcripts were significantly blunted in the HMB group. Mitochondrial respiration and markers of content were both reduced and H2O2 emission was elevated in both groups following BR. CONCLUSIONS: In summary, 10 days of BR in older adults causes a significant deterioration in mitochondrial energetics, while transcriptomic profiling revealed that some of these negative effects may be attenuated by an ONS containing HMB.

Metabolic analysis.

Analysis of the metabolome with coverage of all of the possibly detectable components in the sample, rather than analysis of each individual metabolite at a given time, can be accomplished by metabolic analysis. Targeted and/or nontargeted approaches are applied as needed for particular experiments. Monitoring hundreds or more metabolites at a given time requires high-throughput and high-end techniques that enable screening for relative changes in, rather than absolute concentrations of, compounds within a wide dynamic range. Most of the analytical techniques useful for these purposes use GC or HPLC/UPLC separation modules coupled to a fast and accurate mass spectrometer. GC separations require chemical modification (derivatization) before analysis, and work efficiently for the small molecules. HPLC separations are better suited for the analysis of labile and nonvolatile polar and nonpolar compounds in their native form. Direct infusion and NMR-based techniques are mostly used for fingerprinting and snap phenotyping, where applicable. Discovery and validation of metabolic biomarkers are exciting and promising opportunities offered by metabolic analysis applied to biological and biomedical experiments. We have demonstrated that GC-TOF-MS, HPLC/UPLC-RP-MS and HILIC-LC-MS techniques used for metabolic analysis offer sufficient metabolome mapping providing researchers with confident data for subsequent multivariate analysis and data mining.

Application of liquid chromatography-mass spectrometry analysis in metabolomics: reversed-phase monolithic capillary chromatography and hydrophilic chromatography coupled to electrospray ionization-mass spectrometry.

Analysis of the entire metabolome as the sum of all detectable components in the sample rather than analysis of each individual metabolite is performed by the metabolomics approaches. To monitor in parallel hundreds or even thousands of metabolites, high-throughput techniques are required that enable screening for relative changes rather than absolute concentrations of compounds. Most analytical techniques for profiling small molecules consist of gas chromatography (GC) or high-performance liquid chromatography (HPLC) coupled to mass spectrometry. HPLC separations are better suited for the analysis of labile and high molecular weight compounds, and for the analysis of nonvolatile polar compounds in their natural form. Although GC- and HPLC-based profiling techniques are not truly quantitative, the compounds detecting and employing the acceptable standards may compare their relative amounts. We have demonstrated that reversed-phase monolithic capillary chromatography and hydrophilic chromatography can be successfully applied for sufficient plant crude extracts separations and metabolomics studies.

Further Evaluation of Mechanisms Associated with the Antidepressantlike Signature of Scopolamine in Mice.

BACKGROUND: Conventional antidepressants lack efficacy for many patients (treatmentresistant depression or TRD) and generally take weeks to produce full therapeutic response in others. Emerging data has identified certain drugs such as ketamine as rapidly-acting antidepressants for major depressive disorder and TRD. Scopolamine, a drug used to treat motion sickness and nausea, has also been demonstrated to function as a rapidly-acting antidepressant. The mechanisms associated with efficacy in TRD patients and rapid onset of action have been suggested to involve a-Amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid (AMPA) receptor and mammalian target of rapamycin (mTOR) signaling. Since the work on these mechanisms with scopolamine has been limited, the present set of experiments was designed to further explore these mechanisms of action. METHOD: Male, NIH Swiss mice demonstrated a robust and immediate antidepressant signature with ketamine or scopolamine when studied under the forced-swim test. RESULTS: The AMPA receptor antagonist NBQX prevented this antidepressant-like effect of scopolamine and ketamine. An orally-bioavilable mTOR inhibitor (AZD8055) also attenuated the antidepressant- like effects of scopolamine and ketamine. Scopolamine was also shown to augment the antidepressant- like effect of the selective serotonin reuptake inhibitor citalopram. When given in combination, scopolamine and ketamine acted synergistically to produce antidepressant-like effects. Although drug interaction data suggested that additional mechanisms might be at play, metabolomic analysis of frontal cortex and plasma from muscarinic M1+/+ and M1 -/- mice given scopolamine or vehicle did not reveal any hints as to the nature of these additional mechanisms of action. CONCLUSION: Overall, the data substantiate and extend the idea that AMPA and mTOR signaling pathways are necessary for the antidepressant-like effects of scopolamine and ketamine, mechanisms that appear to be of general significance for TRD therapeutic agents.

A comprehensive workflow of mass spectrometry-based untargeted metabolomics in cancer metabolic biomarker discovery using human plasma and urine.

Current available biomarkers lack sensitivity and/or specificity for early detection of cancer. To address this challenge, a robust and complete workflow for metabolic profiling and data mining is described in details. Three independent and complementary analytical techniques for metabolic profiling are applied: hydrophilic interaction liquid chromatography (HILIC-LC), reversed-phase liquid chromatography (RP-LC), and gas chromatography (GC). All three techniques are coupled to a mass spectrometer (MS) in the full scan acquisition mode, and both unsupervised and supervised methods are used for data mining. The univariate and multivariate feature selection are used to determine subsets of potentially discriminative predictors. These predictors are further identified by obtaining accurate masses and isotopic ratios using selected ion monitoring (SIM) and data-dependent MS/MS and/or accurate mass MSn ion tree scans utilizing high resolution MS. A list combining all of the identified potential biomarkers generated from different platforms and algorithms is used for pathway analysis. Such a workflow combining comprehensive metabolic profiling and advanced data mining techniques may provide a powerful approach for metabolic pathway analysis and biomarker discovery in cancer research. Two case studies with previous published data are adapted and included in the context to elucidate the application of the workflow.

Probing genetic algorithms for feature selection in comprehensive metabolic profiling approach.

Six different clones of 1-year-old loblolly pine (Pinus taeda L.) seedlings grown under standardized conditions in a green house were used for sample preparation and further analysis. Three independent and complementary analytical techniques for metabolic profiling were applied in the present study: hydrophilic interaction chromatography (HILIC-LC/ESI-MS), reversed-phase liquid chromatography (RP-LC/ESI-MS), and gas chromatography all coupled to mass spectrometry (GC/TOF-MS). Unsupervised methods, such as principle component analysis (PCA) and clustering, and supervised methods, such as classification, were used for data mining. Genetic algorithms (GA), a multivariate approach, was probed for selection of the smallest subsets of potentially discriminative classifiers. From more than 2000 peaks found in total, small subsets were selected by GA as highly potential classifiers allowing discrimination among six investigated genotypes. Annotated GC/TOF-MS data allowed the generation of a small subset of identified metabolites. LC/ESI-MS data and small subsets require further annotation. The present study demonstrated that combination of comprehensive metabolic profiling and advanced data mining techniques provides a powerful metabolomic approach for biomarker discovery among small molecules. Utilizing GA for feature selection allowed the generation of small subsets of potent classifiers.

Analysis of highly polar compounds of plant origin: combination of hydrophilic interaction chromatography and electrospray ion trap mass spectrometry.

The primary goal of metabolomic analysis is the unbiased relative quantification of every metabolite in a biological system. A number of different metabolite-profiling techniques must be combined to make this possible. Here we report the separation and analysis of highly polar compounds in a proof of concept study. Compounds were separated and analyzed using hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization (ESI) mass spectrometry. Two types of HILIC microbore columns (Polyhydroxyethyl A and TSK Gel Amide 80) were compared to normal phase silica HPLC columns. The best separations of standards mixtures and plant samples were achieved using the Amide 80 stationary phase. ESI enabled the detection of both positively and negatively charged metabolites, when coupled to a quadrupole ion trap mass spectrometer using continuous polarity switching. By stepwise mass spectrometric fragmentation of the most intense ions, unknown compounds could be identified and then included into a custom mass spectrometric library. This method was used to detect oligosaccharides, glycosides, amino sugars, amino acids, and sugar nucleotides in phloem exudates from petioles of fully expanded Cucurbita maxima leaves. Quantitative analysis was performed using external standards. The detection limit for stachyose was 0.5 ng per injection (Amide 80). The concentration of stachyose in investigated phloem samples was in the range of 1-7 mM depending on the plant.

Monolithic silica-based capillary reversed-phase liquid chromatography/electrospray mass spectrometry for plant metabolomics.

Application of C18 monolithic silica capillary columns in HPLC coupled to ion trap mass spectrometry detection was studied for probing the metabolome of the model plant Arabidopsis thaliana. It could be shown that the use of a long capillary column is an easy and effective approach to reduce ionization suppression by enhanced chromatographic resolution. Several hundred peaks could be detected using a 90-cm capillary column for LC separation and a noise reduction and automatic peak alignment software, which outperformed manual inspection or commercially available mass spectral deconvolution software.