Cross-species comparison of genes related to nutrient sensing mechanisms expressed along the intestine.

INTRODUCTION: Intestinal chemosensory receptors and transporters are able to detect food-derived molecules and are involved in the modulation of gut hormone release. Gut hormones play an important role in the regulation of food intake and the control of gastrointestinal functioning. This mechanism is often referred to as "nutrient sensing". Knowledge of the distribution of chemosensors along the intestinal tract is important to gain insight in nutrient detection and sensing, both pivotal processes for the regulation of food intake. However, most knowledge is derived from rodents, whereas studies in man and pig are limited, and cross-species comparisons are lacking. AIM: To characterize and compare intestinal expression patterns of genes related to nutrient sensing in mice, pigs and humans. METHODS: Mucosal biopsy samples taken at six locations in human intestine (n = 40) were analyzed by qPCR. Intestinal scrapings from 14 locations in pigs (n = 6) and from 10 locations in mice (n = 4) were analyzed by qPCR and microarray, respectively. The gene expression of glucagon, cholecystokinin, peptide YY, glucagon-like peptide-1 receptor, taste receptor T1R3, sodium/glucose cotransporter, peptide transporter-1, GPR120, taste receptor T1R1, GPR119 and GPR93 was investigated. Partial least squares (PLS) modeling was used to compare the intestinal expression pattern between the three species. RESULTS AND CONCLUSION: The studied genes were found to display specific expression patterns along the intestinal tract. PLS analysis showed a high similarity between human, pig and mouse in the expression of genes related to nutrient sensing in the distal ileum, and between human and pig in the colon. The gene expression pattern was most deviating between the species in the proximal intestine. Our results give new insights in interspecies similarities and provide new leads for translational research and models aiming to modulate food intake processes in man.

Instrument and process independent binning and baseline correction methods for liquid chromatography-high resolution-mass spectrometry deconvolution.

Setting appropriate bin sizes to aggregate hyphenated high-resolution mass spectrometry data, belonging to similar mass over charge (m/z) channels, is vital to metabolite quantification and further identification. In a high-resolution mass spectrometer when mass accuracy (ppm) varies as a function of molecular mass, which usually is the case while reading m/z from low to high values, it becomes a challenge to determine suitable bin sizes satisfying all m/z ranges. Similarly, the chromatographic process within a hyphenated system, like any other controlled processes, introduces some process driven systematic behavior that ultimately distorts the mass chromatogram signal. This is especially seen in liquid chromatogram-mass spectrometry (LC-MS) measurements where the gradient of the solvent and the washing step cycle-part of the chromatographic process, produce a mass chromatogram with a non-uniform baseline along the retention time axis. Hence prior to any automatic signal decomposition techniques like deconvolution, it is a equally vital to perform the baseline correction step for absolute metabolite quantification. This paper will discuss an instrument and process independent solution to the binning and the baseline correction problem discussed above, seen together, as an effective pre-processing step toward liquid chromatography-high resolution-mass spectrometry (LC-HR-MS) data deconvolution.

Equating, or correction for between-block effects with application to body fluid LC-MS and NMR metabolomics data sets.

Combination of data sets from different objects (for example, from two groups of healthy volunteers from the same population) that were measured on a common set of variables (for example, metabolites or peptides) is desirable for statistical analysis in "omics" studies because it increases power. However, this type of combination is not directly possible if nonbiological systematic differences exist among the individual data sets, or "blocks". Such differences can, for example, be due to small analytical changes that are likely to accumulate over large time intervals between blocks of measurements. In this article we present a data transformation method, that we will refer to as "quantile equating", which per variable corrects for linear and nonlinear differences in distribution among blocks of semiquantitative data obtained with the same analytical method. We demonstrate the successful application of the quantile equating method to data obtained on two typical metabolomics platforms, i.e., liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. We suggest uni- and multivariate methods to evaluate similarities and differences among data blocks before and after quantile equating. In conclusion, we have developed a method to correct for nonbiological systematic differences among semiquantitative data blocks and have demonstrated its successful application to metabolomics data sets.

Uniform procedure of (1)H NMR analysis of rat urine and toxicometabonomics Part II: comparison of NMR profiles for classification of hepatotoxicity.

A procedure of nuclear magnetic resonance (NMR) urinalysis using pattern recognition is proposed for early detection of toxicity of investigational compounds in rats. The method is applied to detect toxicity upon administration of 13 toxic reference compounds and one nontoxic control compound (mianserine) in rats. The toxic compounds are expected to induce necrosis (bromobenzene, paracetamol, carbon tetrachloride, iproniazid, isoniazid, thioacetamide), cholestasis (alpha-naphthylisothiocyanate (ANIT), chlorpromazine, ethinylestradiol, methyltestosterone, ibuprofen), or steatosis (phenobarbital, tetracycline). Animals were treated daily for 2 or 4 days except for paracetamol and bromobenzene (1 and 2 days) and carbon tetrachloride (1 day only). Urine was collected 24 h after the first and second treatment. The animals were sacrificed 24 h after the last treatment, and NMR data were compared with liver histopathology as well as blood and urine biochemistry. Pathology and biochemistry showed marked toxicity in the liver at high doses of bromobenzene, paracetamol, carbon tetrachloride, ANIT, and ibuprofen. Thioacetamide and chlorpromazine showed less extensive changes, while the influences of iproniazid, isoniazid, phenobarbital, ethinylestradiol, and tetracycline on the toxic parameters were marginal or for methyltestosterone and mianserine negligible. NMR spectroscopy revealed significant changes upon dosing in 88 NMR biomarker signals preselected with the Procrustus Rotation method on principal component discriminant analysis (PCDA) plots. Further evaluation of the specific changes led to the identification of biomarker patterns for the specific types of liver toxicity. Comparison of our rat NMR PCDA data with histopathological changes reported in humans and/or rats suggests that rat NMR urinalysis can be used to predict hepatotoxicity.

Sensitivity of (1)H NMR analysis of rat urine in relation to toxicometabonomics. Part I: dose-dependent toxic effects of bromobenzene and paracetamol.

(1)H nuclear magnetic resonance (NMR) spectroscopy of rat urine in combination with pattern recognition analysis was evaluated for early noninvasive detection of toxicity of investigational chemical entities. Bromobenzene (B) and paracetamol (P) were administered at five single oral dosages between 2 and 500 mg/kg and between 6 and 1800 mg/kg, respectively. The sensitivity of the proposed method to detect changes in the NMR spectra 24 and 48 h after single dosing was compared with histopathology and biochemical parameters in plasma and urine. Both B and P applied at the highest dosages induced liver necrosis and markedly increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) plasma levels. At dosages of 125 mg/kg B and 450 mg/kg P, liver necrosis and changes in AST and ALT were less pronounced, while at lower dose levels these effects could not be detected. Changes in kidney pathology or standard urine biochemistry were not observed at any of these dosages. Evaluation of the total NMR dataset showed 80 signals to be sensitive for B and P dosing. Principal component analysis on the reduced dataset revealed that NMR spectra were significantly different at dosages above 8 mg/kg (B) and 110 mg/kg (P) at both sampling times. This implies a 4- to 16-fold increased sensitivity of NMR versus histopathology and clinical chemistry in recognizing early events of liver toxicity.

Integrative biological analysis of the APOE*3-leiden transgenic mouse.

Integrative (or systems biology) is a new approach to analyzing biological entities as integrated systems of genetic, genomic, protein, metabolite, cellular, and pathway events that are in flux and interdependent. Here, we demonstrate the application of intregrative biological analysis to a mammalian disease model, the apolipoprotein E3-Leiden (APO*E3) transgenic mouse. Mice selected for the study were fed a normal chow diet and sacrificed at 9 weeks of age-conditions under which they develop only mild type I and II atherosclerotic lesions. Hepatic mRNA expression analysis showed a 25% decrease in APO A1 and a 43% increase in liver fatty acid binding protein expression between transgenic and wild type control mice, while there was no change in PPAR-alpha expression. On-line high performance liquid chromatography-mass spectrometry quantitative profiling of tryptic digests of soluble liver proteins and liver lipids, coupled with principle component analysis, enabled rapid identification of early protein and metabolite markers of disease pathology. These included a 44% increase in L-FABP in transgenic animals compared to controls, as well as an increase in triglycerides and select bioactive lysophosphatidylcholine species. A correlation analysis of identified genes, proteins, and lipids was used to construct an interaction network. Taken together, these results indicate that integrative biology is a powerful tool for rapid identification of early markers and key components of pathophysiologic processes, and constitute the first application of this approach to a mammalian system.

Methods for the differential integrative omic analysis of plasma from a transgenic disease animal model.

Multitiered quantitative analysis of biological systems is rapidly becoming the desired approach to study hierarchical functional interactions between proteins and metabolites. We describe here a novel systematic approach to analyze organisms with complex metabolic regulatory networks. By using precise analytical methods to measure biochemical constituents and their relative abundance in whole plasma of transgenic ApoE*3-Leiden mice and an isogenic wild-type control group, simultaneous snapshots of metabolic and protein states were obtained. Novel data processing and multivariate analysis tools such as Impurity Resolution Software (IMPRESS) and Windows-based linear fit program (WINLIN) were used to compare protein and metabolic profiles in parallel. Canonical correlations of the resulting data show quantitative relationships between heterogeneous components in the TG animals. These results, obtained solely from whole plasma analysis allowed us, in a rapid manner, to corroborate previous findings as well as find new events pertaining to dominant and peripheral events in lipoprotein metabolism of a genetically modified mammalian organism in relation to ApoE3, a key mediator of lipoprotein metabolism.

1H-NMR spectroscopy combined with pattern recognition analysis reveals characteristic chemical patterns in urines of MS patients and non-human primates with MS-like disease.

Proton nuclear magnetic resonance (1H-NMR) spectroscopy in combination with pattern recognition techniques were used to investigate the composition of organic compounds in urines from patients with multiple sclerosis (MS), patients with other neurological diseases (OND) and healthy controls (H). Using a valid animal model of MS, namely the common marmoset (Callithrix jacchus) model of experimental autoimmune encephalomyelitis (EAE), the relation of disease progression and alteration of the urine composition was investigated. Urine samples were collected during different stages of EAE, either induced with whole human myelin or with the myelin protein MOG in complete adjuvant. The urine samples were analysed with 1H-NMR spectroscopy allowing simultaneous detection of an array of compounds. Spectral differences between urines from EAE-affected and healthy monkeys were assessed with multivariate analysis. Evidence is provided that development of EAE is associated with changes in the chemical composition of the urine, in particular of compounds with NMR peaks in the region of the spectrum between 0.5 and 3.50 ppm. In addition, we found preliminary evidence for differences between urines from MS, OND and H groups.

Identification of disease- and nutrient-related metabolic fingerprints in osteoarthritic Guinea pigs.

Osteoarthritis (OA), one of the most common diseases among the elderly, is characterized by the progressive destruction of joint tissues. Its etiology is largely unclear and no effective disease-modifying treatment is currently available. Metabolic fingerprinting provides a novel tool for the identification of biomarkers. A metabolic fingerprint consists of a typical combination of metabolites in a biological fluid and is identified by a combination of (1)H NMR spectroscopy and multivariate data analysis (MVDA). The current feasibility study was aimed at identifying a metabolic fingerprint for OA and applying this in a nutritional intervention study. Urine samples were collected from osteoarthritic male Hartley guinea pigs (n = 44) at 10 and 12 mo of age, treated from 4 mo onward with variable vitamin C doses (2.5-3, 30 and 150 mg/d) and from healthy male Strain 13 guinea pigs (n = 8) at 12 mo of age, treated with 30 mg vitamin C/d. NMR measurements were performed on all urine samples. Subsequently, MVDA was carried out on the data obtained using NMR. An NMR fingerprint was identified that reflected the osteoarthritic changes in guinea pigs. The metabolites that comprised the fingerprint indicate that energy and purine metabolism are of major importance in OA. Metabolic fingerprinting also allowed detection of differences in OA-specific metabolites induced by different dietary vitamin C intakes. This study demonstrates the feasibility of metabolic fingerprinting to identify disease-specific profiles of urinary metabolites. NMR fingerprinting is a promising means of identifying new disease markers and of gaining fresh insights into the pathophysiology of disease.