Label-free mass spectrometric profiling of urinary proteins and metabolites from paediatric idiopathic nephrotic syndrome.

Idiopathic nephrotic syndrome (INS) is caused by renal diseases that increase the permeability of the glomerular filtration barrier without evidence of a specific systemic cause. The aim of the present work was to reveal inherent molecular features of INS in children using combined urinary proteomics and metabolomics profiling. In this study, label-free mass spectrometric analysis of urinary proteins and small molecule metabolites was carried out in 12 patients with INS versus 12 sex- and age-matched control subjects with normal renal function. Integration and biological interpretation of obtained results were carried out by Ingenuity IPA software. Validation of obtained proteomics data was carried out by Western blot method. Proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000765. This study indicates for the first time that paediatric INS is associated with up-regulation of afamin, hydroxyphenylacetate and uridine, and concomitant down-regulation in glutamine and phenylalanine levels, and many of these molecular species were previously shown to be involved in oxidative stress. Further studies in larger patient population are underway to investigate the role of oxidative stress in renal injury in paediatric INS.

Strategy for optimizing LC-MS data processing in metabolomics: a design of experiments approach.

A strategy for optimizing LC-MS metabolomics data processing is proposed. We applied this strategy on the XCMS open source package written in R on both human and plant biology data. The strategy is a sequential design of experiments (DoE) based on a dilution series from a pooled sample and a measure of correlation between diluted concentrations and integrated peak areas. The reliability index metric, used to define peak quality, simultaneously favors reliable peaks and disfavors unreliable peaks using a weighted ratio between peaks with high and low response linearity. DoE optimization resulted in the case studies in more than 57% improvement in the reliability index compared to the use of the default settings. The proposed strategy can be applied to any other data processing software involving parameters to be tuned, e.g., MZmine 2. It can also be fully automated and used as a module in a complete metabolomics data processing pipeline.

Systematic evaluation of extraction methods for multiplatform-based metabotyping: application to the Fasciola hepatica metabolome.

Combining data from multiple analytical platforms is essential for comprehensive study of the molecular phenotype (metabotype) of a given biological sample. The metabolite profiles generated are intrinsically dependent on the analytical platforms, each requiring optimization of instrumental parameters, separation conditions, and sample extraction to deliver maximal biological information. An in-depth evaluation of extraction protocols for characterizing the metabolome of the hepatobiliary fluke Fasciola hepatica , using ultra performance liquid chromatography and capillary electrophoresis coupled with mass spectroscopy is presented. The spectrometric methods were characterized by performance, and metrics of merit were established, including precision, mass accuracy, selectivity, sensitivity, and platform stability. Although a core group of molecules was common to all methods, each platform contributed a unique set, whereby 142 metabolites out of 14,724 features were identified. A mixture design revealed that the chloroform:methanol:water proportion of 15:59:26 was globally the best composition for metabolite extraction across UPLC-MS and CE-MS platforms accommodating different columns and ionization modes. Despite the general assumption of the necessity of platform-adapted protocols for achieving effective metabotype characterization, we show that an appropriately designed single extraction procedure is able to fit the requirements of all technologies. This may constitute a paradigm shift in developing efficient protocols for high-throughput metabolite profiling with more-general analytical applicability.

Use of an atmospheric solids analysis probe (ASAP) for high throughput screening of biological fluids: preliminary applications on urine and bile.

A hybrid quadrupole orthogonal time-of-flight mass spectrometer (QToF) equipped with a solids analysis probe (atmospheric solids analysis probe-mass spectrometry (ASAP-MS)) has been applied to the high throughput qualitative analysis of bile (rat and dog) and urine (rat) samples. The metabolic profiles generated by ASAP-MS was less comprehensive than that provided by liquid chromatography (LC) or gas chromatography-mass spectrometry (GC-MS) metabonomic profiling, though simple types of sample preparation were found to increase the range of ions detected for bile (a complex, multicompartment sample type). While unsuited to biomarker discovery, ASAP-MS of these biofluids generated sufficiently complex metabolic fingerprints to enable them to be distinguished from each other using multivariate statistical methods such as principal components analysis (PCA). This ability to provide an effective means of sample classification suggests possible diagnostic applications.

Comprehensive LC-MS E lipidomic analysis using a shotgun approach and its application to biomarker detection and identification in osteoarthritis patients.

A fast and robust method for lipid profiling utilizing liquid chromatography coupled with mass spectrometry has been demonstrated and validated for the analysis of human plasma. This method allowed quantification and identification of lipids in human plasma using parallel alternating low energy and high energy collision spectral acquisition modes. A total of 275 [corrected] lipids were identified and quantified (as relative concentrations) in both positive and negative ion electrospray ionization mode. The method was validated with five nonendogenous lipids, and the linearity (r(2) better than 0.994) and the intraday and interday repeatability (relative standard deviation, 4-6% and 5-8%, respectively) were satisfactory. The developed lipid profiling method was successfully applied for the analysis of plasma from osteoarthritis (OA) patients. The multivariate statistical analysis by partial least-squares-discrimination analysis suggested an altered lipid metabolism associated with osteoarthritis and the release of arachidonic acid from phospholipids.

Perturbations in fatty acid metabolism and apoptosis are manifested in calcific coronary artery disease: An exploratory lipidomic study.

BACKGROUND: Controversy exists concerning the beneficial or harmful effects of the presence of ectopic calcification in the coronary arteries. Additionally, further elucidation of the exact pathophysiological mechanism is needed. In this study, we sought to identify metabolic markers of vascular calcification that could assist in understanding the disease, monitoring its progress and generating hypotheses describing its pathophysiology. METHODS: Untargeted lipid profiling and complementary modeling strategies were employed to compare serum samples from patients with different levels of calcific coronary artery disease (CCAD) based on their calcium score (CS). Subsequently, patients were divided into three groups: no calcification (NC; CS=0; n=26), mild calcification (MC; CS:1-250; n=27) and severe (SC; CS>250; n=17). RESULTS: Phosphatidylcholine levels were found to be significantly altered in the disease states (p=0.001-0.04). Specifically, 18-carbon fatty acyl chain (FAC) phosphatidylcholines were detected in lower levels in the SC group, while 20:4 FAC lipid species were detected in higher concentrations. A statistical trend was observed with phosphatidylcholine lipids in the MC group, showing the same tendency as with the SC group. We also observed several sphingomyelin signals present at lower intensities in SC when compared with NC or MC groups (p=0.000001-0.01). CONCLUSIONS: This is the first lipid profiling study reported in CCAD. Our data demonstrate dysregulations of phosphatidylcholine lipid species, which suggest perturbations in fatty acid elongation/desaturation. The altered levels of the 18-carbon and 20:4 FAC lipids may be indicative of disturbed inflammation homeostasis. The marked sphingomyelin dysregulation in SC is consistent with profound apoptosis as a potential mechanism of CCAD.

Metabonomic applications in toxicity screening and disease diagnosis.

Biofluid NMR spectroscopy is a powerful tool providing a comprehensive metabolic profile of the low molecular weight components in biofluids that reflect concentrations and fluxes of endogenous metabolites involved in key intermediary cellular pathways, thereby giving an indication of an organisms physiological or pathophysiological status [1]. The interaction of pharmacological agents with cells and tissues can also be monitored using recently developed high resolution magic-angle spinning (HRMAS) NMR spectroscopic technology for biological matrices [1]. However, recent developments in both spectrometer and software technology has resulted in improved capacity for sample handling, leading to a rapid growth in the size of toxicological spectral databases, and increased the complexity of the biological spectral data generated. Thus more emphasis has been placed on the need to develop improved automated procedures for data processing and interpretation. By harnessing chemometric tools for analysis of complex spectral data, the toxicological consequences of xenobiotic exposure can be evaluated efficiently on line. Automation of spectral processing procedures and the construction of mathematically based 'expert systems' for the prediction of drug-induced toxicity founded on IH NMR spectral profiles have now been achieved. Chemometric analysis of biological NMR spectra has provided the main analytical platform for metabonomic analysis, providing a systems approach to evaluating pathophysiological or genetic influences on the metabolic status of an organism [1]. This technology is currently being given high-priority in the pharmaceutical industry with respect to development of efficient high throughput toxicity screening systems for lead candidate selection. In this article, we review the recent developments in metabonomics and consider their application in toxicological screening, disease diagnosis and functional genomics.

The lipid composition of isolated cytoplasmic lipid droplets from a human cancer cell line, BE(2)M17.

(1)H nuclear magnetic resonance spectroscopy (NMR) resonances from lipids in tumours are associated with tumour grade and treatment response. The origin of these NMR signals is mainly considered to be cytoplasmic lipid droplets (LDs). Techniques exist for isolating LDs but little is known about their composition and its relationship to NMR signals. In this work, density-gradient ultracentrifugation was performed on homogenised human cancer cells to isolate LDs. (1)H NMR was performed on whole cells, isolated LDs and their extracts. Heteronuclear single quantum coherence spectroscopy (HSQC) and liquid chromatography mass spectroscopy (LC-MS) were performed on lipid extracts of LDs. Staining and microscopy were used to characterize isolated LDs. An excellent agreement in chemical shift and relative signal intensity was observed between lipid resonances in cells and isolated LD spectra supporting that NMR-visible lipids originate primarily from LDs. Isolated LDs showed high concentrations of unsaturated lipids, a oleic-to-linoleic acid ratio greater than two and a cholesteryl ester (ChE)-to-cholesterol (Ch) ratio close to unity. These ratios were several-fold greater than respective ratios in whole cells, demonstrating isolation is important to characterize LD composition. LDs contain a specific group of lipid species that are likely to contribute to the (1)H NMR spectrum of cells.

H NMR spectroscopy-based metabolomic assessment of uremic toxicity, with toxicological outcomes, in male rats following an acute, mid-life insult from ochratoxin a.

Overt response to a single 6.25 mg dose of ochratoxin A (OTA) by oral gavage to 15 months male rats was progressive loss of weight during the following four days. Lost weight was restored within one month and animals had a normal life-span without OTA-related terminal disease. Decline in plasma OTA concentration only commenced four days after dosing, while urinary excretion of OTA and ochratoxin alpha was ongoing. During a temporary period of acute polyuria, a linear relationship between urine output and creatinine concentration persisted. Elimination of other common urinary solutes relative to creatinine was generally maintained during the polyuria phase, except that phosphate excretion increased temporarily. (1)H NMR metabolomic analysis of urine revealed a progressive cyclic shift in the group principal components data cluster from before dosing, throughout the acute insult phase, and returning almost completely to normality when tested six months later. Renal insult by OTA was detected by (1)H NMR within a day of dosing, as the most sensitive early indicator. Notable biomarkers were trimethylamine N-oxide and an aromatic urinary profile dominated by phenylacetylglycine. Tolerance of such a large acute insult by OTA, assessed by rat natural lifetime outcomes, adds a new dimension to toxicology of this xenobiotic.

Generic dealkylation: a tool for increasing the hit-rate of metabolite rationalization, and automatic customization of mass defect filters.

The use of exact mass liquid chromatography/mass spectrometry (LC/MS) for drug metabolism studies has increased significantly in recent years. Firstly, exact mass measurements facilitate identification of standard biotransformations through the use of narrow window extracted ion chromatograms, which are typically highly selective relative to signals from matrix or dosing components. Secondly, novel metabolites can be characterized via elemental formula calculations and high-resolution product ion spectra. Furthermore, biological background ions can be removed by the use of mass defect filters (MDFs) which filter out ions based on the decimal component of their m/z value. Here, we describe an approach which we term 'generic dealkylation' that in association with other data interpretation tools adds significant value to the assignment process. Generic dealkylation uses a simple strategy to identify those bonds which have the potential to be cleaved by metabolism. In combination with standard phase 1 and phase 2 biotransformations, this allows creation of a chemically intelligent MDF which balances the need to remove matrix background with the requirement of avoiding filtering true metabolites. Secondly, generic dealkylation increases the hit-rate at which non-trivial (i.e. not covered by simple phase 1 oxidations or direct phase 2 conjugations) metabolites can be directly rationalized. The value of the generic dealkylation approach is illustrated by its application to determination of in vitro metabolic routes for two commercial drugs, nefazodone and indinavir.

Visualization of GC/TOF-MS-based metabolomics data for identification of biochemically interesting compounds using OPLS class models.

Metabolomics studies generate increasingly complex data tables, which are hard to summarize and visualize without appropriate tools. The use of chemometrics tools, e.g., principal component analysis (PCA), partial least-squares to latent structures (PLS), and orthogonal PLS (OPLS), is therefore of great importance as these include efficient, validated, and robust methods for modeling information-rich chemical and biological data. Here the S-plot is proposed as a tool for visualization and interpretation of multivariate classification models, e.g., OPLS discriminate analysis, having two or more classes. The S-plot visualizes both the covariance and correlation between the metabolites and the modeled class designation. Thereby the S-plot helps identifying statistically significant and potentially biochemically significant metabolites, based both on contributions to the model and their reliability. An extension of the S-plot, the SUS-plot (shared and unique structure), is applied to compare the outcome of multiple classification models compared to a common reference, e.g., control. The used example is a gas chromatography coupled mass spectroscopy based metabolomics study in plant biology where two different transgenic poplar lines are compared to wild type. By using OPLS, an improved visualization and discrimination of interesting metabolites could be demonstrated.

Heteronuclear 19F-1H statistical total correlation spectroscopy as a tool in drug metabolism: study of flucloxacillin biotransformation.

We present a novel application of the heteronuclear statistical total correlation spectroscopy (HET-STOCSY) approach utilizing statistical correlation between one-dimensional 19F/1H NMR spectroscopic data sets collected in parallel to study drug metabolism. Parallel one-dimensional (1D) 800 MHz 1H and 753 MHz 19F{1H} spectra (n = 21) were obtained on urine samples collected from volunteers (n = 6) at various intervals up to 24 h after oral dosing with 500 mg of flucloxacillin. A variety of statistical relationships between and within the spectroscopic datasets were explored without significant loss of the typically high 1D spectral resolution, generating 1H-1H STOCSY plots, and novel 19F-1H HET-STOCSY, 19F-19F STOCSY, and 19F-edited 1H-1H STOCSY (X-STOCSY) spectroscopic maps, with a resolution of approximately 0.8 Hz/pt for both nuclei. The efficient statistical editing provided by these methods readily allowed the collection of drug metabolic data and assisted structure elucidation. This approach is of general applicability for studying the metabolism of other fluorine-containing drugs, including important anticancer agents such as 5-fluorouracil and flutamide, and is extendable to any drug metabolism study where there is a spin-active X-nucleus (e.g., 13C, 15N, 31P) label present.

Novel application of reversed-phase UPLC-oaTOF-MS for lipid analysis in complex biological mixtures: a new tool for lipidomics.

Ultra-Performance LC (UPLC) utilizing sub-2-mum porous stationary phase particles operating with high linear velocities at pressures >9000 psi was coupled with orthogonal acceleration time-of-flight (oaTOF) mass spectrometry and successfully employed for the rapid separation of lipids from complex matrices. The UPLC system produced information-rich chromatograms with typical measured peak widths of 3 s at peak base, generating peak capacities in excess of 200 in 10 min. Further UPLC coupled with MSE technology provided parent and fragment mass information of lipids in one chromatographic run, thus, providing an attractive alternative to current LC methods for targeted lipid analysis as well as lipidomic studies.

MSE with mass defect filtering for in vitro and in vivo metabolite identification.

Metabolite identification studies involve the detection and structural characterization of the biotransformation products of drug candidates. These experiments are necessary throughout the drug discovery and development process. The use of high-resolution chromatography and high-resolution mass spectrometry together with data processing using mass defect filtering is described for in vitro and in vivo metabolite identification studies. Data collection was done using UPLC coupled with an orthogonal hybrid quadrupole time-of-flight mass spectrometer. This experimental approach enabled the use of MS(E) data collection (where E represents collision energy) which has previously been shown to be a powerful approach for metabolite identification studies. Post-acquisition processing with a prototype mass defect filtering program was used to eliminate endogenous interferences in the study samples, greatly enhancing the discovery of metabolites. The ease of this approach is illustrated by results showing the detection and structural characterization of metabolites in plasma from a preclinical rat pharmacokinetic study.

The detection of phenotypic differences in the metabolic plasma profile of three strains of Zucker rats at 20 weeks of age using ultra-performance liquid chromatography/orthogonal acceleration time-of-flight mass spectrometry.

Analysis of biological fluids using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) (metabonomics) can allow new insights to be gained into disease processes, with advances in chromatographic techniques enabling the detection of thousands of metabolites. In this work metabonomics has been used to investigate the metabolic processes involved in type II diabetes in the Zucker obese rat. Plasma was analyzed from three different strains, the Zucker (fa/fa) obese, Zucker lean and the lean/(fa) obese cross. Using UPLC/MS, ca. 10,000 ions were detected due to the narrow peak widths and excellent peak shapes achieved with this technology. Confidence in the chromatographic performance was demonstrated by the use of quality control standards. The positive and negative ion total ion chromatograms obtained from the three strains were readily distinguishable using multivariate statistical analysis. The greatest difference was observed between the Zucker lean and Zucker lean/(fa) rats compared to the Zucker (fa/fa) obese rats. Positive ions m/z 220 (4.36 min), 282(3.78 min), 359 (5.33 min) and 405 (7.77 min) were elevated in the plasma derived from Zucker lean rats whilst ions m/z 385 (6.80 min) and 646 (4.36 min) were at a lower concentration compared to the plasma from the Zucker (fa/fa) obese animals. Negative ions elevated in the Zucker lean rats included m/z 212 (2.30 min), 514 (2.85 min), 295 (4.39 min), 329 (3.11 min), 343 (2.86 min) and 512 (2.86 min) with ions m/z 538 (4.18 min), 568 (4.18 min), 568 (5.09 min) and 612 (4.30 min) being raised in the samples derived from Zucker (fa/fa) obese animals. The ion m/z 514 (3.85 min) was found to correspond to taurocholate, providing further support for an involvement of taurine metabolism in diabetes.

Comparative metabonomics of differential hydrazine toxicity in the rat and mouse.

Interspecies variation between rats and mice has been studied for hydrazine toxicity using a novel metabonomics approach. Hydrazine hydrochloride was administered to male Sprague-Dawley rats (30 mg/kg, n = 10 and 90 mg/kg, n = 10) and male B6C3F mice (100 mg/kg, n = 8 and 250 mg/kg, n = 8) by oral gavage. In each species, the high dose was selected to produce the major histopathologic effect, hepatocellular lipid accumulation. Urine samples were collected at sequential time points up to 168 h post dose and analyzed by 1H NMR spectroscopy. The metabolites of hydrazine, namely diacetyl hydrazine and 1,4,5,6-tetrahydro-6-oxo-3-pyridazine carboxylic acid (THOPC), were detected in both the rat and mouse urine samples. Monoacetyl hydrazine was detected only in urine samples from the rat and its absence in the urine of the mouse was attributed to a higher activity of N-acetyl transferases in the mouse compared with the rat. Differential metabolic effects observed between the two species included elevated urinary beta-alanine, 3-D-hydroxybutyrate, citrulline, N-acetylcitrulline, and reduced trimethylamine-N-oxide excretion unique to the rat. Metabolic principal component (PC) trajectories highlighted the greater degree of toxic response in the rat. A data scaling method, scaled to maximum aligned and reduced trajectories (SMART) analysis, was used to remove the differences between the metabolic starting positions of the rat and mouse and varying magnitudes of effect, to facilitate comparison of the response geometries between the rat and mouse. Mice followed "biphasic" open PC trajectories, with incomplete recovery 7 days after dosing, whereas rats followed closed "hairpin" time profiles, indicating functional reversibility. The greater magnitude of metabolic effects observed in the rat was supported by the more pronounced effect on liver pathology in the rat when compared with the mouse.