Urine metabolomics is widely used for biomarker research in the fields of medicine and toxicology. As a consequence, characterization of the variations of the urine metabolome under basal conditions becomes critical in order to avoid confounding effects in cohort studies. Such physiological information is however very scarce in the literature and in metabolomics databases so far. Here we studied the influence of age, body mass index (BMI), and gender on metabolite concentrations in a large cohort of 183 adults by using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). We implemented a comprehensive statistical workflow for univariate hypothesis testing and modeling by orthogonal partial least-squares (OPLS), which we made available to the metabolomics community within the online Workflow4Metabolomics.org resource. We found 108 urine metabolites displaying concentration variations with either age, BMI, or gender, by integrating the results from univariate p-values and multivariate variable importance in projection (VIP). Several metabolite clusters were further evidenced by correlation analysis, and they allowed stratification of the cohort. In conclusion, our study highlights the impact of gender and age on the urinary metabolome, and thus it indicates that these factors should be taken into account for the design of metabolomics studies.

To get more insight into plant cell response to cadmium (Cd) stress, both proteomic and metabolomic "differential display" analyses were performed on Arabidopsis thaliana cells exposed to different concentrations of the toxic chemical. After a 24 h treatment, soluble proteins extracted from untreated and treated cells were separated by 2-D-PAGE and image analyses were performed to quantify and compare protein levels. Proteins up- and down-regulated in response to Cd were identified by MS and mapped into specific metabolic pathways and cellular processes, highlighting probable activation of the carbon, nitrogen, and sulfur metabolic pathways. For some of these proteins, Northern blot and RT-PCR analyses were performed to test transcript accumulation in response to Cd. In parallel, metabolite profiling analyses by LC coupled to ESI MS were initiated to better characterize the metabolic adaptation to the chemical stress. This study revealed that the main variation at the metabolite level came from the presence of six different families of phytochelatins, in A. thaliana cells treated with Cd, whose accumulation increases with Cd concentrations. Taken together these data provide an overview of the molecular and cellular changes elicited by Cd exposure.

Angiotensin I-converting enzyme (ACE) has two homologous active NH 2 -and COOH-terminal domains and displays activity toward a broad range of substrates. The tetrapeptide N -acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has been shown to be hydrolyzed in vitro by ACE and to be a preferential substrate for its NH 2 -terminal active site. This peptide is a regulatory factor of hematopoiesis which reversibly prevents the recruitment of pluripotent hematopoietic stem cells and normal early progenitors into S-phase. We found that a single oral dose of 50 mg of the ACE inhibitor, captopril, when administered to eight healthy subjects in a double-blind, crossover, placebo-controlled study, massively increased the plasma level of Ac-SDKP.

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Recently, high-resolution mass spectrometry has been largely employed for compound identification, thanks to accurate mass measurements. As additional information, relative isotope abundance (RIA) is often needed to reduce the number of candidates prior to tandem MS(n). Here, we report on the evaluation of the LTQ-Orbitrap, in terms of accurate mass and RIA measurements for building further metabolomics spectral databases. Accurate mass measurements were achieved in the ppm range, using external calibration within 24 h, and remained at <5 ppm over a one-week period. The experimental relative abundances of (M+1) isotopic ions were evaluated in different data sets. First of all, 137 solutions of commercial compounds were analyzed by flow injection analysis in both the positive and negative ion modes. It was found that the ion abundance was the main factor impacting the accuracy of RIA measurements. It was possible to define some intensity thresholds above which errors were systematically <20% of their theoretical values. The same type of results were obtained with analyses from two biological media. Otherwise, no significant effect of ion transmission between the LTQ ion trap and the Orbitrap analyzer on RIA measurement errors was found, whereas the reliability of RIA measurements was dramatically improved by reducing the mass detection window. It was also observed that the signal integration method had a significant impact on RIA measurement errors, with the most-reliable results being obtained with peak height integrations. Finally, automatic integrations using the data preprocessing software XCMS and MZmine gave results similar to those obtained by manual integration, suggesting that it is relevant to use the RIA information in automatic elemental composition determination software from metabolomic peak tables.

IntroductionThe renin-angiotensin system plays a critical role in blood pressure regulation and fluid hemodynamics. Pharmacologic inhibitors of this system are routinely used to treat hypertension and congestive heart failure. One of the most controversial effects of the reninangiotensin system has been the interplay of this system with erythrocyte production. A variety of clinical reports have noted an association between activation of the renin-angiotensin system and increased erythropoiesis (1-3). These studies have come from analyses of patients with a variety of chronic diseases including chronic obstructive pulmonary disease, heart failure, and renal transplantation. Other investigators have suggested a link between angiotensin-converting enzyme (ACE) inhibitors and worsened anemia, particularly in patients with chronic renal failure (4-6). While research has focused on the interplay of the renin-angiotensin system and erythropoietin, no mechanistic explanation for these observations has been generally accepted.Central to the renin-angiotensin system is ACE, a peptidase that converts angiotensin I to angiotensin II (7). In mammals, most ACE is bound to tissues such as endothelium, but enzymatic cleavage results in a circulating form within plasma. In vitro, ACE is capable of cleaving many small peptides besides angiotensin I. However, in vivo, with the exception of bradykinin, the significance of nonangiotensin peptides as ACE substrates is not well understood. ACE is a protein with two independent catalytic domains. While both catalytic sites hydrolyze angiotensin I with roughly equal efficiency, the amino-and carboxy-terminal catalytic domains differ in their rate constants for other peptides.Using targeted homologous recombination in embryonic stem (ES) cells, our laboratory created two lines of mice with modifications of the ACE gene (8, 9). These animals are termed ACE.1 and ACE.2. Mice homozygous for the ACE.1 allele (ACE.1 knockout mice) are null for all ACE production. They have a marked reduction of blood pressure, and a renal lesion characterized by hypoplasia of the renal medulla and papilla. In contrast to this null phenotype, animals homozygous for the ACE.2 allele (ACE.2 knockout mice) have a partial restoration of ACE activity. These animals express a truncated ACE protein containing only the amino-terminal catalytic domain. Since this shortened ACE protein lacks the carboxy-terminal domain that normally anchors ACE to cell membranes, the ACE.2 protein is exported from cells into blood and other extracellular fluids. Thus, while the plasma of ACE.2 mice converts angiotensin I to angiotensin II with about 34% of the activity of wild-type mouse plasma, tissues such as the lung and kidney completely lack ACE protein or activity. The systolic blood pressure of ACE.2 knockout mice averaged 75 mmHg, as low as that of the ACE.1 knockout animals.Here, we investigate an unexpected finding concerning the phenotypes of both the ACE.1 and ACE.2 mice. These animals are anemic. ACE.2 knockout mice are a p...

Metabolic profiles of biofluids obtained by atmospheric pressure ionization mass spectrometry-based technologies contain hundreds to thousands of features, most of them remaining unknown or at least not characterized in analytical systems. We report here on the annotation of the human adult urinary metabolome and metabolite identification from electrospray ionization mass spectrometry (ESI-MS)based metabolomics data sets. Features of biological interest were first of all annotated using the ESI-MS database of the laboratory. They were also grouped, thanks to software tools, and annotated using public databases. Metabolite identification was achieved using two complementary approaches: (i) formal identification by matching chromatographic retention times, mass spectra, and also product ion spectra (if required) of metabolites to be characterized in biological data sets to those of reference compounds and (ii) putative identification from biological data thanks to MS/MS experiments for metabolites not available in our chemical library. By these means, 384 metabolites corresponding to 1484 annotated features (659 in negative ion mode and 825 in positive ion mode) were characterized in human urine samples. Of these metabolites, 192 and 66 were formally and putatively identified, respectively, and 54 are reported in human urine for the first time. These lists of features could be used by other laboratories to annotate their ESI-MS metabolomics data sets.

Monitoring molecular dynamics of an organism upon stress is probably the best approach to decipher physiological mechanisms involved in the stress response. Quantitative analysis of proteins and metabolites is able to provide accurate information about molecular changes allowing the establishment of a range of more or less specific mechanisms, leading to the identification of major players in the considered pathways. Such tools have been successfully used to analyze the plant response to cadmium (Cd), a major pollutant capable of causing severe health issues as it accumulates in the food chain. We present a summary of proteomics and metabolomics works that contributed to a better understanding of the molecular aspects involved in the plant response to Cd. This work allowed us to provide a finer picture of general signaling, regulatory and metabolic pathways that appeared to be affected upon Cd stress. In particular, we conclude on the advantage of employing different approaches of global proteome- and metabolome-wide techniques, combined with more targeted analysis to answer molecular questions and unravel biological networks. Finally, we propose possible directions and methodologies for future prospectives in this field, as many aspects of the plant-Cd interaction remain to be discovered.