A metabonomic analysis of plasma from Zucker rat strains using gas chromatography/mass spectrometry and pattern recognition

Plasma obtained from three strains of Zucker rats was analysed using capillary gas chromatography/mass spectrometry (GC/MS) to obtain global metabolite profiles as part of a series of metabonomic investigations of animal models of diabetes. Samples were obtained from 20-week-old male wild-type Zucker lean, (fa/fa) obese and lean/(fa) animals and were analysed following protein precipitation, using acetonitrile, and derivatisation. Subsequent data analysis using principal components analysis (PCA) and orthogonal projection to latent structures (OPLS) revealed differences between the plasma metabolite profiles of the three strains, with those of the Zucker lean and the lean/(fa) crosses being similar to each other whilst differing from the (fa/fa) obese strain.     

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.     

Profiling and biomarker identification in plasma from different Zucker rat strains via high mass accuracy multistage mass spectrometric analysis using liquid chromatography/mass spectrometry with a quadrupole ion trap-time of flight mass spectrometer

High mass accuracy electrospray ionisation multistage tandem mass spectrometry (MS(n)) was applied to metabolite profiling studies on plasma samples derived from two strains of rat (the Zucker (fa/fa) obese strain and the normal wild type). Using a quadrupole ion trap time-of-flight (QIT-TOF) mass spectrometer, metabolite profiling software was applied to locate components of biological significance that could account for the differences between the two strains of rat and a formula prediction software tool was used to help identify individual components. The primary factor discriminating between the two populations was the concentration of endogenous lipids. In the Zucker (fa/fa) obese strain, the dominant ion signals and MS(n) spectra were in agreement with lysoglycerophosphocholine components such as palmitoyllysophosphatidylcholine, 1-oleoylglycerophosphocholine, 1-octadecyl-sn-glycero-3-phosphocholine and 1-stearoylglycerophosphocholine and these were found in relatively higher concentrations compared to the normal wild type. Components were identified using high mass accuracy MS(n) data, formula prediction software and by agreement with published mass spectra through internet databases, rather than using a conventional approach with authentic standards. This application shows that the use of high mass accuracy electrospray ionisation MS(n) together with a software tool can be used effectively to detect and characterise unknown analytes in complex matrices, and represents a promising approach for future profiling studies.     

Beneficial Effect of Quercetin on Erythrocyte Properties in Type 2 Diabetic Rats

Diabetes mellitus is characterized by tissue oxidative damage and impaired microcirculation, as well as worsened erythrocyte properties. Measurements of erythrocyte deformability together with determination of nitric oxide (NO) production and osmotic resistance were used for the characterization of erythrocyte functionality in lean (control) and obese Zucker diabetic fatty (ZDF) rats of two age categories. Obese ZDF rats correspond to prediabetic (younger) and diabetic (older) animals. As antioxidants were suggested to protect erythrocytes, we also investigated the potential effect of quercetin (20 mg/kg/day for 6 weeks). Erythrocyte deformability was determined by the filtration method and NO production using DAF-2DA fluorescence. For erythrocyte osmotic resistance, we used hemolytic assay. Erythrocyte deformability and NO production deteriorated during aging—both were lower in older ZDF rats than in younger ones. Three-way ANOVA indicates improved erythrocyte deformability after quercetin treatment in older obese ZDF rats only, as it was not modified or deteriorated in both (lean and obese) younger and older lean animals. NO production by erythrocytes increased post treatment in all experimental groups. Our study indicates the potential benefit of quercetin treatment on erythrocyte properties in condition of diabetes mellitus. In addition, our results suggest potential age-dependency of quercetin effects in diabetes that deserve additional research.     

Obesity aggravates the joint inflammation in a collagen-induced arthritis model through deviation to Th17 differentiation

White fat cells secrete adipokines that induce inflammation and obesity has been reported to be characterized by high serum levels of inflammatory cytokines such as IL-6 and TNF-α. Rheumatoid arthritis (RA) is a prototype of inflammatory arthritis, but the relationship between RA and obesity is controversial. We made an obese inflammatory arthritis model: obese collagen-induced arthritis (CIA). C57BL/6 mice were fed a 60-kcal high fat diet (HFD) from the age of 4 weeks and they were immunized twice with type II collagen (CII). After immunization, the obese CIA mice showed higher arthritis index scores and histology scores and a more increased incidence of developing arthritis than did the lean CIA mice. After treatment with CII, mixed lymphocyte reaction also showed CII-specific response more intensely in the obese CIA mice than lean CIA. The anti-CII IgG and anti-CII IgG2a levels in the sera of the obese CIA mice were higher than those of the lean CIA mice. The number of Th17 cells was higher and the IL-17 mRNA expression of the splenocytes in the obese CIA mice was higher than that of the lean CIA mice. Obese CIA mice also showed high IL-17 expression on synovium in immunohistochemistry. Although obesity may not play a pathogenic role in initiating arthritis, it could play an important role in amplifying the inflammation of arthritis through the Th1/Th17 response. The obese CIA murine model will be an important tool when we investigate the effect of several therapeutic target molecules to treat RA.     

Bee Bread Can Alleviate Lipid Abnormalities and Impaired Bone Morphology in Obese Zucker Diabetic Rats

This study examined for the first time whether bee bread (BB, consisting of monofloral rape bee pollen) could alleviate lipid derangements and reduced bone quality in Zucker diabetic fatty (ZDF) rats, which are considered an appropriate animal model for type 2 diabetes mellitus (T2DM) investigation. Adult ZDF rats were segregated into four groups: lean non-diabetic rats (L group), obese diabetic rats untreated (C group), and those treated with the BB at two doses (500 and 700 mg/kg body weight, respectively, B1 and B2 groups) for 10 weeks. Significantly reduced levels of total cholesterol and triglyceride were recorded in the B2 group versus the C group. In both BB-treated groups, significantly increased relative volume of trabecular bone and trabecular thickness, enhanced density of secondary osteons, accelerated periosteal bone apposition, and improved blood flow were observed. A positive effect of higher dose of BB on femoral weight and cortical bone thickness was also demonstrated. Our results suggest a promising potential of BB to ameliorate T2DM-related complications associated with lipid and bone damages.     

Metabolite identification of small interfering RNA duplex by high-resolution accurate mass spectrometry

On-line liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using an LTQ-Orbitrap mass spectrometer was employed to investigate the metabolite profiles of a model siRNA duplex designated HBV263. The HBV263 duplex was incubated in rat and human serum and liver microsomes in vitro. The siRNA drug and its metabolites were then extracted using a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE), and analyzed by LC/ESI-MS. High-resolution accurate mass data enabled differentiation between two possible metabolite sequences with a monoisotopic molecular mass difference of less than 1 Da. ProMass deconvolution software was used to provide semi-automated data processing. In vitro serum and liver microsome incubation samples afforded different metabolite patterns: the antisense strand of the duplex was degraded preferentially in rat and human serum, while the sense strand of the duplex was less stable in rat and human liver microsomes.     

Characterization of metabolites of meisoindigo in male and female rat kidney microsomes by high-performance liquid chromatography coupled with positive electrospray ionization tandem mass spectrometry

Meisoindigo has been effectively applied for the treatment of chronic myelogenous leukemia (CML). Although the metabolic profile of meisoindigo has been studied in liver, information relevant to extrahepatic metabolism of meisoindigo is absent in kidney so far. In this study, the metabolism of meisoindigo in rat kidney microsomes was qualitatively and quantitatively investigated by liquid chromatography/tandem mass spectrometry (LC/MS/MS), in terms of metabolite identification, metabolic stability, metabolite formation and gender effect. The metabolic profiling was accomplished by integration of multiple reaction monitoring (MRM) with conventional full MS scan followed by MS/MS methodology. The major in vitro metabolites of meisoindigo in rat kidney microsomes were identified as stereoselective 3,3' double-bond reduced meisoindigo, whereas the minor metabolites were regioselective phenyl monohydroxylmeisoindigo. An LC/MS/MS method for quantification of meisoindigo in rat kidney microsomes was also developed and validated. The calculated in vitro half-life (t(1/2)) values of meisoindigo in male and female rat kidney microsomes were 107.8 +/- 17.0 min and 130.0 +/- 12.9 min, respectively. There were no statistically significant differences between different genders in the metabolic stability profiles of meisoindigo. The reductive metabolite-formation profiles of meisoindigo in male and female rat kidney microsomes were plotted semi-quantitatively as well. The information regarding in vitro renal metabolism of meisoindigo provided a better understanding of the role of the kidney in the disposition of meisoindigo.     

Metabolite profiles of epimedin C in rat plasma and bile by ultra‐performance liquid chromatography coupled with quadrupole‐TOF‐MS

Epimedin C is one of the major bioactive constituents of Herba Epimedii. In this study, the metabolite profiles of epimedin C in rat plasma and bile were qualitatively investigated, and the possible metabolic pathways of epimedin C were subsequently proposed. After oral administration of epimedin C at a single dose of 80 mg/kg, rat biological samples were collected and pretreated by protein precipitation. Then these pretreated samples were injected into an Acquity UPLC BEH C₁₈ column and detected by ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry. In all, 12 metabolites were identified in the biosamples. Of these, eight, two from plasma and six from bile, are, to our knowledge, reported here for the first time. The results indicated that epimedin C was metabolized via desugarization, dehydrogenation, hydrogenation, dehydroxylation, hydroxylation, demethylation and glucuronidation pathways in vivo. Thus, this study revealed the possible metabolite profiles of epimedin C in rat plasma and bile. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of pharmacokinetics,bioavailability and urinary excretion of scopolin and its metabolite scopoletin in Sprague Dawley rats by liquid chromatography–tandem mass spectrometry

We aimed to investigate the pharmacokinetics, bioavailability and urinary excretion of scopolin and its metabolite scopoletin in rats. An LC–tandem mass spectrometry (MS/MS) method for simultaneous determination of scopolin and scopoletin in rat biomatrices was developed and validated over a plasma and urine concentration range of 5.0–2000 ng/mL. Chromatographic separation was performed on a Hypersil GOLD C₁₈ column with acetonitrile and 0.1% formic acid in water as mobile phase with gradient elution. Detection was performed in the positive ionization and selected reaction monitoring mode. The intra‐ and inter‐batch precision and accuracy, extraction recovery and matrix effect and stability of scopolin and scopoletin were well within the acceptable limits of variation. There was no gender‐related difference in the pharmacokinetic profiles of scopolin. There were significant differences in total area under the concentration–time curve (AUC), time required to achieve a maximal concentration (T_max) and apparent clearance from plasma (Cl/F) of scopoletin between the male and female rats (p < .05). The bioavailability (F) of scopolin was exceptionally low. The maximal excretion rates were 7.61 μg/h and 7.15 μg/h for scopolin and 31.68 μg/h and 25.58 μg/h for scopoletin in male and female rats, respectively. The LC–MS/MS method was successfully applied to the pharmacokinetic, bioavailability and urinary excretion studies of scopolin and its metabolite scopoletin following a single administration of scopolin to rats.     

Biochemical and Molecular Investigation of the Effect of Saponins and Terpenoids Derived from Leaves of Ilex aquifolium on Lipid Metabolism of Obese Zucker Rats

Ilex paraguariensis, the holly tree, is a plant with recognized biological properties, whose aqueous infusions are known as “Yerba mate”, that regulate lipid metabolism, reduce obesity, and improve brain stimulation. In the present study, the effect of standardized saponin and terpenoid fractions of a European taxon, Ilex aquifolium, on blood biochemical parameters in a rat model of metabolic disorder, (fa/fa) Zucker, are presented. The profiles of the volatile fractions of two species and six European varieties of Ilex were investigated. After selecting the best variety, the saponin and terpenoid fractions were isolated and standardized, and animals were fed 10 mg kg⁻¹ b.w. for 8 weeks. A statistically significant decrease in liver adiposity was observed, confirmed by histology and quantitative identification (gas chromatography–mass spectrometry analyses of hepatic lipids. RT-qPCR analysis of gene expression in the aorta revealed that the administration of the terpenoid fraction downregulated LOX-1, suggesting a reduction in atherosclerotic stimuli. In addition, a statistically significant reduction (p < 0.05) in PPARγ for the saponin fraction was observed in the liver. The expression of the ACAT-1 gene in the liver, responsible for the formation of cholesterol esters, increased significantly in the group receiving the terpenoid fraction compared to the control, which was also confirmed by the analysis of individual blood biochemical parameters. The opposite effect was observed for saponins. Taking the above into account, it is shown for the first time that Ilex aquifolium can be a source of compounds that positively influence lipid metabolism.     

Seed coat metabolite profiling of cowpea (Vigna unguiculata L. Walp.) accessions from Ghana using UPLC-PDA-QTOF-MS and chemometrics

Abstract

Cowpea (Vigna unguiculata L. Walp.) is an important grain legume in Africa exhibiting high morpho-genetic diversity. However, not much information exists on the phytochemical profiles of its hulls. This study explored the metabolite profiles of seed-coats from thirteen cowpea accessions of varying phenotypes using UPLC-QTOF-MS and chemometric analysis. A total of 34 secondary metabolites were identified, which comprised phenolic acids, flavonoids, anthocyanins, sphingolipids and fatty acids. Quantification of selected phenolic compounds revealed marked variations among the cowpea accessions. The chemical profiles of the test accessions were distinguished by multivariate analysis, and the results revealed a marked influence of seed-coat pigmentation on the observed differences in their metabolite profiles. Moreover, delphinidin (traces to 2257.6 µg/g), catechin glucoside (traces to 2840.6 µg/g), catechin (traces to 2089.2 µg/g) and epicatechin (26.3 to 3222.7 µg/g) contributed to the segregation amongst the studied samples. The concentrations of the discriminant metabolites were greater in the dark seeded cowpeas compared to their lighter seeded counterparts. The findings represent a useful contribution to the literature on cowpea seed coat metabolites, and also reveal their potential for use in the development of food and pharmaceutical products.     

Interspecies in vitro metabolism of the phosphodiesterase-4 (PDE4) inhibitor L-454,560

L-454,560 is a potent phosphodiesterase 4 (PDE4) inhibitor which was identified as a development candidate for the treatment of asthma and chronic obstructive pulmonary disease (COPD). As part of the discovery of this compound, interspecies in vitro metabolism data was generated using liver microsomes and hepatocytes in order to understand the metabolic fate of the compound. In microsomes, metabolism of the 3-methyl-1,2,4-oxadiazole ring was the predominant pathway observed, including ring cleavage. In rat hepatocytes, hydroxylation of the methyl group on the oxadiazole ring and double-bond isomerization were the most abundant metabolites observed. No major species differences were found in terms of microsomal metabolite profiles. The use of LC with UV and MS detection is highlighted, as well as information from tandem mass spectrometry and NMR.     

Is metabolomics reachable? Different purification strategies of human colon cancer cells provide different CE-MS metabolite profiles

In this work, four different metabolite purification approaches are investigated prior to metabolomics of human HT29 colon cancer cells. Namely, methanol deproteinization, ultrafiltration and two SPE methods using C18 and polymer-based cartridges were studied. The extracts were characterized via a metabolomic approach based on the application of CE TOF MS (CE-MS). CE-MS analysis time was less than 20 min per sample and allowed the simultaneous and reproducible analysis of more than 80 metabolites in a single run with a minimum consumption of sample and reagents. Metabolome analysis revealed in some cases important differences among the studied metabolite purification procedures. No significant differences were observed in the metabolite profile using C18 and polymer-based cartridges, or between ultrafiltration and methanol deproteinization. However, important differences were observed in the metabolomic profiles obtained from SPE and methanol deproteinization samples. These results demonstrate the crucial role of the metabolite purification strategy in metabolomics since it can bias (and in some cases mislead) the conclusions achieved by the metabolomic study.     

The role of molecular chaperonins in warm ischemia and reperfusion injury in the steatotic liver: A proteomic study

ABSTRACT: BACKGROUND: The molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software. RESULTS: The relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1was significantly upregulated in steatotic liver following I/R. CONCLUSION: Down-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.     

Monte Carlo efficiency calibration of a neutron generator-based total-body irradiator

Many body composition measurement systems are calibrated against a single-sized reference phantom. Prompt-gamma neutron activation (PGNA) provides the only direct measure of total body nitrogen (TBN), an index of the body’s lean tissue mass. In PGNA systems, body size influences neutron flux attenuation, induced gamma signal distribution, and counting efficiency. Thus, calibration based on a single-sized phantom could result in inaccurate TBN values. We used Monte Carlo simulations (MCNP-5; Los Alamos National Laboratory) in order to map a system’s response to the range of body weights (65–160 kg) and body fat distributions (25–60%) in obese humans. Calibration curves were constructed to derive body-size correction factors relative to a standard reference phantom, providing customized adjustments to account for differences in body habitus of obese adults. The use of MCNP-generated calibration curves should allow for a better estimate of the true changes in lean tissue mass that many occur during intervention programs focused only on weight loss.     

Discovering disease-associated enzymes by proteome reactivity profiling

Proteomics aims to identify new markers and targets for the diagnosis and treatment of human disease. To realize this goal, methods and reagents are needed to profile proteins based on their functional properties, rather than mere abundance. Here, we describe a general strategy for synthesizing and evaluating structurally diverse libraries of activity-based proteomic probes. Quantitative screening of probe-proteome reactions coupled with bioinformatic analysis enabled the selection of a suite of probes that exhibit complementary protein reactivity profiles. This optimal probe set was applied to discover several enzyme activities differentially expressed in lean and obese (ob/ob) mice. Interestingly, one of these enzymes, hydroxypyruvate reductase, which was 6-fold upregulated in ob/ob livers, participates in the conversion of serine to glucose, suggesting that this unusual metabolic pathway may contribute to gluconeogenesis selectively in states of obesity.     

大鼠肝脏纤维化病变的核磁共振研究

利用高分辨魔角旋转核磁共振技术(HR-MAS NMR)以及主成分分析法(PCA)对大鼠肝脏组织纤维化病变前后以及不同程度病变的代谢产物变化进行了研究. 结果表明, 纤维化病变使得肝脏脂肪含量增加; 葡萄糖、糖原、牛磺酸以及肌醇的含量相对减小. 相对磷酸胆碱(PC)而言, 胆碱(Cho)及甘油磷酸胆碱(GPC)降低. 随着正常至纤维化病变程度的加深, 脂肪含量有一个先显著上升, 再缓慢下降的趋势; 而不同程度纤维化病变阶段之间其它代谢产物的变化不明显. 主成分分析证实了上述的结果, 正常与纤维化病变样本中代谢产物的组成存在较大的差异性, 可被明显分为两组; 而各纤维化病变阶段之间的差异不明显.     

Metabolite Variation in Lean and Obese Streptozotocin (STZ)-Induced Diabetic Rats via <Superscript>1</Superscript>H NMR-Based Metabolomics Approach

Diabetes mellitus (DM) is considered as a complex metabolic disease because it affects the metabolism of glucose and other metabolites. Although many diabetes studies have been conducted in animal models throughout the years, the pathogenesis of this disease, especially between lean diabetes (ND + STZ) and obese diabetes (OB + STZ), is still not fully understood. In this study, the urine from ND + STZ, OB + STZ, lean/control (ND), and OB + STZ rats were collected and compared by using ¹H NMR metabolomics. The results from multivariate data analysis (MVDA) showed that the diabetic groups (ND + STZ and OB + STZ) have similarities and dissimilarities for a certain level of metabolites. Differences between ND + STZ and OB + STZ were particularly noticeable in the synthesis of ketone bodies, branched-chain amino acid (BCAA), and sensitivity towards the oral T2DM diabetes drug metformin. This finding suggests that the ND + STZ group was more similar to the T1DM model and OB + STZ to the T2DM model. In addition, we also managed to identify several pathways and metabolism aspects shared by obese (OB) and OB + STZ. The results from this study are useful in developing drug target-based research as they can increase understanding regarding the cause and effect of DM.     

Study of the in vitro metabolism of TJ0711 using ultra high performance liquid chromatography with quadrupole time‐of‐flight and ultra fast liquid chromatography with quadrupole linear ion trap mass spectrometry

TJ0711 (1‐[4‐(2‐methoxyethyl)phenoxy]‐3‐[2‐(2‐methoxyphenoxy)ethylamino]‐2‐propanol) is a novel β‐adrenoreceptor blocker with vasodilating activity. The aim of this study was to investigate the in vitro metabolic properties of TJ0711 from both qualitative and quantitative aspects using mouse, rat, dog, and human liver microsomes as well as rat hepatocytes. Two modern liquid chromatography with tandem mass spectrometry systems, ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry and ultra fast liquid chromatography with quadrupole linear ion trap mass spectrometry, were utilized for the analysis. To better characterize the metabolic pathways of TJ0711, two major metabolites were incubated under the same conditions as that for TJ0711. TJ0711 was extensively metabolized in vitro, and a total of 34 metabolites, including 19 phase I and 15 phase II metabolites, were identified. Similar metabolite profiles were observed among species, and demethylation, hydroxylation, carboxylic acid formation, and glucuronidation were proposed as the major metabolic routes. Significant interspecies differences were observed in the metabolic stability studies of TJ0711. Furthermore, gender differences were significant in mice, rats, and dogs, but were negligible in humans. The valuable information provided in this work will be useful in planning and interpreting further pharmacokinetic, in vivo metabolism and toxicological studies of this novel β‐blocker.