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The following study investigates the preparation of human blood plasma for metabolomic profiling analysis by ultrahigh performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC/TOFMS) in a novel two-step design study. Four different organic solvents (acetonitrile, acetone, methanol, and ethanol) were used to assess human blood plasma preparation via protein precipitation. The optimal conditions for sample preparation were investigated, with consideration to the number of extracted markers, data quality/reproducibility, and column lifetime prolongation. Isotopically labeled internal standards were used to monitor data quality/reproducibility. Gel electrophoresis was also used to measure the protein content in the supernatant of the 'first design step' allowing assessment of the amount of protein that would be injected/accumulate onto the column after many injections that would be apparent in a global metabolic profiling study. The second design step followed on from the results obtained in step one, with four of the best conditions selected and further investigated, looking at the effects of vortex time and temperature on precipitation/extraction. Two choices of solvent compositions were found to be 'optimal' for preparation of plasma for global metabolic profiling analysis; these were 'methanol/ethanol' (1:1, v/v) and 'methanol/acetonitrile/acetone' (1: 1:1, v/v/v) added to plasma (4:1 ratio, 400 [micro]L total volume).

A new analytical strategy for biomarker recovery from directly coupled ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC Tof MS) data on biofluids is presented and exemplified using a study on hydrazine-induced liver toxicity. A key step in the strategy involves a novel procedure for reducing the spectroscopic search space by differential analysis of cohorts of normal and pathological samples using an orthogonal projection to latent structures discriminant analysis (O-PLS-DA). This efficiently sorts principal discriminators of toxicity from the background of thousands of metabolic features commonly observed in data sets generated by UPLC--MS analysis of biological fluids and is thus a powerful tool for biomarker discovery.

Statistical heterospectroscopy (SHY) is a new statistical paradigm for the coanalysis of multispectroscopic data sets acquired on multiple samples. This method operates through the analysis of the intrinsic covariance between signal intensities in the same and related molecules measured by different techniques across cohorts of samples. The potential of SHY is illustrated using both 600-MHz [sup.1]H NMR and UPLC-TOFMS data obtained from control rat urine samples (n = 54) and from a corresponding hydrazine-treated group (n = 58). We show that direct cross-correlation of spectral parameters, viz. chemical shifts from NMR and m/z data from MS, is readily achievable for a variety of metabolites, which leads to improved efficiency of molecular biomarker identification. In addition to structure, higher level biological information can be obtained on metabolic pathway activity and connectivities by examination of different levels of the NMR to MS correlation and anticorrelation matrixes. The SHY approach is of general applicability to complex mixture analysis, if two or more independent spectroscopic data sets are available for any sample cohort. Biological applications of SHY as demonstrated here show promise as a new systems biology tool for biomarker recovery.

Abstract Background Expression profiling of embryonic stem (ES) cell differentiation in the presence of serum has been performed previously. It remains unclear if transcriptional activation is dependent on complex growth factor mixtures in serum or whether this process is intrinsic to ES cells once the stem cell program has been inactivated. The aims of this study were to determine the transcriptional programs associated with the stem cell state and to characterize mesoderm differentiation between serum and serum-free culture. Results ES cells were differentiated as embryoid bodies in 10% FBS or serum-free media containing BMP4 (2 ng/ml), and expression profiled using 47 K Illumina(R) Sentrix arrays. Statistical methods were employed to define gene sets characteristic of stem cell, epiblast and primitive streak programs. Although the initial differentiation profile was similar between the two culture conditions, cardiac gene expression was inhibited in serum whereas blood gene expression was enhanced. Also, expression of many members of the Kruppel-like factor (KLF) family of transcription factors changed dramatically during the first few days of differentiation. KLF2 and KLF4 co-localized with OCT4 in a sub-nuclear compartment of ES cells, dynamic changes in KLF-DNA binding activities occurred upon differentiation, and strong bio-informatic evidence for direct regulation of many stem cell genes by KLFs was found. Conclusion Down regulation of stem cell genes and activation of epiblast/primitive streak genes is similar in serum and defined media, but subsequent mesoderm differentiation is strongly influenced by the composition of the media. In addition, KLF family members are likely to be important regulators of many stem cell genes.

RATIONALE The aim of the work was to develop and validate a method for the quantification of vitamin D metabolites in serum using ultra-high-pressure liquid chromatography coupled to mass spectrometry (LC/MS), and to validate a high-resolution mass spectrometry (LC/HRMS) approach against a tandem mass spectrometry (LC/MS/MS) approach using a large clinical sample set. METHODS A fast, accurate and reliable method for the quantification of the vitamin D metabolites, 25-hydroxyvitamin D2 (25OH-D2) and 25-hydroxyvitamin D3 (25OH-D3), in human serum was developed and validated. The C3 epimer of 25OH-D3 (3-epi-25OH-D3) was also separated from 25OH-D3. The samples were rapidly prepared via a protein precipitation step followed by solid-phase extraction (SPE) using an HLB μelution plate. Quantification was performed using both LC/MS/MS and LC/HRMS systems. RESULTS Recovery, matrix effect, inter- and intra-day reproducibility were assessed. Lower limits of quantification (LLOQs) were determined for both 25OH-D2 and 25OH-D3 for the LC/MS/MS approach (6.2 and 3.4 µg/L, respectively) and the LC/HRMS approach (2.1 and 1.7 µg/L, respectively). A Passing & Bablok fit was determined between both approaches for 25OH-D3 on 662 clinical samples (1.11 + 1.06x). It was also shown that results can be affected by the inclusion of the isomer 3-epi-25OH-D3. CONCLUSIONS Quantification of the relevant vitamin D metabolites was successfully developed and validated here. It was shown that LC/HRMS is an accurate, powerful and easy to use approach for quantification within clinical laboratories. Finally, the results here suggest that it is important to separate 3-epi-25OH-D3 from 25OH-D3. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Alkylresorcinols (AR) are phenolic lipids found in the bran fraction of whole-grain wheat, rye, and barley. In intervention studies, plasma AR concentration increased in response to greater intakes of whole grain, wheat, and rye. This study examined the cross-sectional associations between plasma AR and habitual whole-grain intake, BMI, and metabolic risk factors in 407 free-living older adults (166 men and 241 women; aged 60-81 y; median BMI: 27 kg/m2). Plasma AR were measured by liquid chromatography-tandem MS, and whole-grain intakes were estimated by using an FFQ. After adjustment for fasting TG concentrations, median plasma AR concentrations across quartile categories of AR were 5, 14, 27, and 62 nmol/L, respectively. Spearman correlation coefficients between plasma AR and whole-grain wheat-rich foods and total bran intake were 0.31 and 0.27, respectively (both P < 0.0001). After adjustment for multiple covariates, the geometric means of BMI in the lowest and highest quartile category of plasma AR were 27.6 and 26.7 kg/m2, respectively (P-trend = 0.04). No associations were observed between plasma AR and glucose and insulin. Our study shows a dose-dependent relationship between whole-grain intake and plasma AR and confirms the previously observed inverse relationship between whole-grain intake and BMI using an independent biomarker of whole-grain wheat intake. [ABSTRACT FROM AUTHOR]

Background: Predictive disease risk biomarkers that can be linked to exposure have proved difficult to identify in case-control studies. Methods: Parallel statistical analysis of the correlation between 1H NMR profiles from plasma samples collected before disease onset (EPIC cohort), versus exposure to dietary compounds, and follow-up disease endpoints (colon and breast cancer) was performed. Results: Metabonomic signatures associated with colon cancer and dietary fiber intake (a protective factor according to epidemiological studies) were identified. Conclusion: This implementation of the novel '' meet-in-the-middle'' analytical strategy indicates how case-control studies nested in prospectively collected cohorts may reveal intermediate biomarkers linking exposure and disease. [ABSTRACT FROM AUTHOR]

The article focuses on summary recommendations for standardization and reporting of metabolic analyses. The Standard Metabolic Reporting Structures (SMRS) group is a collection of interested parties from academia, industry and the government that first came together in November 2003 to derive and recommend standards for conducting and reporting metabonomics and metabolomics studies. These multivariate metabolic analyses have diverse applications, including pre-clinical drug safety assessment, disease diagnosis, plant metabolite profiling and environmental science. For a particular algorithm used to derive a model, any specific parameters relevant to the algorithm and the particular implementation of algorithm used should be stated. Because biological research now relies on so many different technologies, various protocols, terms, definitions, methods and reporting structures have developed within sub-disciplines employing the various techniques.

β-Klotho (encoded by Klb) is an obligate coreceptor, mediating both fibroblast growth factor (FGF)15 and FGF21 signaling. Klb -/- mice are refractory to metabolic FGF15 and FGF21 action and exhibit derepressed (increased) bile acid (BA) synthesis. Here, we deeply phenotyped male Klb -/- mice on a pure C57BL/6J genetic background, fed a chow diet focusing on metabolic aspects. This aims to better understand the physiological consequences of concomitant FGF15 and FGF21 signaling deficiency, in particular on the gut-liver axis. Klb -/- mice present permanent growth restriction independent of adiposity and energy balance. Klb -/- mice also exhibit few changes in carbohydrate metabolism, combining normal gluco-tolerance, insulin sensitivity, and fasting response with increased gluconeogenic capacity and decreased glycogen mobilization. Livers of Klb -/- mice reveal pathologic features, including a proinflammatory status and initiation of fibrosis. These defects are associated to a massive shift in BA composition in the enterohepatic system and blood circulation featured by a large excess of microbiota-derived deoxycholic acid, classically known for its genotoxicity in the gastrointestinal tract. In conclusion, β-Klotho is a gatekeeper of hepatic integrity through direct action (mediating FGF21 anti-inflammatory signaling) and indirect mechanisms (mediating FGF15 signaling that maintains BA level and composition).