Ureaplasma parvum : understanding the complexities of intra-amniotic infection in an ovine model

The human Ureaplasma species are the most frequently isolated bacteria from the upper genital tract of pregnant women and can cause clinically asymptomatic, intra-uterine infections, which are difficult to treat with antimicrobials. Ureaplasma infection of the upper genital tract during pregnancy has been associated with numerous adverse outcomes including preterm birth, chorioamnionitis and neonatal respiratory diseases. The mechanisms by which ureaplasmas are able to chronically colonise the amniotic fluid and avoid eradication by (i) the host immune response and (ii) maternally-administered antimicrobials, remain virtually unexplored. To address this gap within the literature, this study investigated potential mechanisms by which ureaplasmas are able to cause chronic, intra-amniotic infections in an established ovine model. In this PhD program of research the effectiveness of standard, maternal erythromycin for the treatment of chronic, intra-amniotic ureaplasma infections was evaluated. At 55 days of gestation pregnant ewes received an intra-amniotic injection of either: a clinical Ureaplasma parvum serovar 3 isolate that was sensitive to macrolide antibiotics (n = 16); or 10B medium (n = 16). At 100 days of gestation, ewes were then randomised to receive either maternal erythromycin treatment (30 mg/kg/day for four days) or no treatment. Ureaplasmas were isolated from amniotic fluid, chorioamnion, umbilical cord and fetal lung specimens, which were collected at the time of preterm delivery of the fetus (125 days of gestation). Surprisingly, the numbers of ureaplasmas colonising the amniotic fluid and fetal tissues were not different between experimentally-infected animals that received erythromycin treatment or infected animals that did not receive treatment (p > 0.05), nor were there any differences in fetal inflammation and histological chorioamnionitis between these groups (p > 0.05). These data demonstrate the inability of maternal erythromycin to eradicate intra-uterine ureaplasma infections. Erythromycin was detected in the amniotic fluid of animals that received antimicrobial treatment (but not in those that did not receive treatment) by liquid chromatography-mass spectrometry; however, the concentrations were below therapeutic levels (<10 – 76 ng/mL). These findings indicate that the ineffectiveness of standard, maternal erythromycin treatment of intra-amniotic ureaplasma infections may be due to the poor placental transfer of this drug. Subsequently, the phenotypic and genotypic characteristics of ureaplasmas isolated from the amniotic fluid and chorioamnion of pregnant sheep after chronic, intra-amniotic infection and low-level exposure to erythromycin were investigated. At 55 days of gestation twelve pregnant ewes received an intra-amniotic injection of a clinical U. parvum serovar 3 isolate, which was sensitive to macrolide antibiotics. At 100 days of gestation, ewes received standard maternal erythromycin treatment (30 mg/kg/day for four days, n = 6) or saline (n = 6). Preterm fetuses were surgically delivered at 125 days of gestation and ureaplasmas were cultured from the amniotic fluid and the chorioamnion. The minimum inhibitory concentrations (MICs) of erythromycin, azithromycin and roxithromycin were determined for cultured ureaplasma isolates, and antimicrobial susceptibilities were different between ureaplasmas isolated from the amniotic fluid (MIC range = 0.08 – 1.0 mg/L) and chorioamnion (MIC range = 0.06 – 5.33 mg/L). However, the increased resistance to macrolide antibiotics observed in chorioamnion ureaplasma isolates occurred independently of exposure to erythromycin in vivo. Remarkably, domain V of the 23S ribosomal RNA gene (which is the target site of macrolide antimicrobials) of chorioamnion ureaplasmas demonstrated significant variability (125 polymorphisms out of 422 sequenced nucleotides, 29.6%) when compared to the amniotic fluid ureaplasma isolates and the inoculum strain. This sequence variability did not occur as a consequence of exposure to erythromycin, as the nucleotide substitutions were identical between chorioamnion ureaplasmas isolated from different animals, including those that did not receive erythromycin treatment. We propose that these mosaic-like 23S ribosomal RNA gene sequences may represent gene fragments transferred via horizontal gene transfer. The significant differences observed in (i) susceptibility to macrolide antimicrobials and (ii) 23S ribosomal RNA sequences of ureaplasmas isolated from the amniotic fluid and chorioamnion suggests that the anatomical site from which they were isolated may exert selective pressures that alter the socio-microbiological structure of the bacterial population, by selecting for genetic changes and altered antimicrobial susceptibility profiles. The final experiment for this PhD examined antigenic size variation of the multiple banded antigen (MBA, a surface-exposed lipoprotein and predicted ureaplasmal virulence factor) in chronic, intra-amniotic ureaplasma infections. Previously defined ‘virulent-derived’ and ‘avirulent-derived’ clonal U. parvum serovar 6 isolates (each expressing a single MBA protein) were injected into the amniotic fluid of pregnant ewes (n = 20) at 55 days of gestation, and amniotic fluid was collected by amniocentesis every two weeks until the time of near-term delivery of the fetus (at 140 days of gestation). Both the avirulent and virulent clonal ureaplasma strains generated MBA size variants (ranging in size from 32 – 170 kDa) within the amniotic fluid of pregnant ewes. The mean number of MBA size variants produced within the amniotic fluid was not different between the virulent (mean = 4.2 MBA variants) and avirulent (mean = 4.6 MBA variants) ureaplasma strains (p = 0.87). Intra-amniotic infection with the virulent strain was significantly associated with the presence of meconium-stained amniotic fluid (p = 0.01), which is an indicator of fetal distress in utero. However, the severity of histological chorioamnionitis was not different between the avirulent and virulent groups. We demonstrated that ureaplasmas were able to persist within the amniotic fluid of pregnant sheep for 85 days, despite the host mounting an innate and adaptive immune response. Pro-inflammatory cytokines (interleukin (IL)-1â, IL-6 and IL-8) were elevated within the chorioamnion tissue of pregnant sheep from both the avirulent and virulent treatment groups, and this was significantly associated with the production of anti-ureaplasma IgG antibodies within maternal sera (p < 0.05). These findings suggested that the inability of the host immune response to eradicate ureaplasmas from the amniotic cavity may be due to continual size variation of MBA surface-exposed epitopes. Taken together, these data confirm that ureaplasmas are able to cause long-term in utero infections in a sheep model, despite standard antimicrobial treatment and the development of a host immune response. The overall findings of this PhD project suggest that ureaplasmas are able to cause chronic, intra-amniotic infections due to (i) the limited placental transfer of erythromycin, which prevents the accumulation of therapeutic concentrations within the amniotic fluid; (ii) the ability of ureaplasmas to undergo rapid selection and genetic variation in vivo, resulting in ureaplasma isolates with variable MICs to macrolide antimicrobials colonising the amniotic fluid and chorioamnion; and (iii) antigenic size variation of the MBA, which may prevent eradication of ureaplasmas by the host immune response and account for differences in neonatal outcomes. The outcomes of this program of study have improved our understanding of the biology and pathogenesis of this highly adapted microorganism.

(Ultra)fast catalyst-free macromolecular conjugation in aqueous environment at ambient temperature

Tailor-made water-soluble macromolecules, including a glycopolymer, obtained by living/controlled RAFT-mediated polymerization are demonstrated to react in water with diene-functionalized poly(ethylene glycol)s without pre- or post-functionalization steps or the need for a catalyst at ambient temperature. As previously observed in organic solvents, hetero-Diels-Alder (HDA) conjugations reached quantitative conversion within minutes when cyclopentadienyl moieties were involved. However, while catalysts and elevated temperatures were previously necessary for open-chain diene conjugation, additive-free HDA cycloadditions occur in water within a few hours at ambient temperature. Experimental evidence for efficient conjugations is provided via unambiguous ESI-MS, UV/vis, NMR, and SEC data.

Challenges in mass spectrometry-based quantification of bioactive peptides: A case study exploring the neuropeptide Y family

The study of biologically active peptides is critical to the understanding of physiological pathways, especially those involved in the development of disease. Historically, the measurement of biologically active endogenous peptides has been undertaken by radioimmunoassay, a highly sensitive and robust technique that permits the detection of physiological concentrations in different biofluid and tissue extracts. Over recent years, a range of mass spectrometric approaches have been applied to peptide quantification with limited degrees of success. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to the NPY family exhibiting regulatory effects on appetite and feeding behavior. The physiological significance of these peptides depends on their molecular forms and in vivo concentrations systemically and at local sites within tissues. In this report, we describe an approach for quantification of individual peptides within mixtures using high-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the NPY family peptides. Aspects of quantification including sample preparation, the use of matrix-matched calibration curves, and internal standards will be discussed. This method for the simultaneous determination of NPY, PYY, and PP was accurate and reproducible but lacks the sensitivity required for measurement of their endogenous concentration in plasma. The advantages of mass spectrometric quantification will be discussed alongside the current obstacles and challenges. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 357–366, 2012.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(l-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/GF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

Studies on the surface properties of biodegradable polymer carriers in respiratory delivery of drug from Dry Powder Inhaler formulations

Dry Powder Inhaler (DPI) technology has a significant impact in the treatment of various respiratory disorders. DPI formulations consist of a micronized drug (<5ìm) blended with an inert coarse carrier, for which lactose is widely used to date. DPIs are one of the inhalation devices which are used to target the delivery of drugs to the lungs. Drug delivery via DPI formulations is influenced by the physico-chemical characteristics of lactose particles such as size, shape, surface roughness and adhesional forces. Commercially available DPI formulations, which utilise lactose as the carrier, are not efficient in delivering drug to the lungs. The reasons for this are the surface morphology, adhesional properties and surface roughness of lactose. Despite several attempts to modify lactose, the maximum efficient drug delivery to the lungs remains limited; hence, exploring suitable alternative carriers for DPIs is of paramount importance. Therefore, the objective of the project was to study the performance of spherical polymer microparticles as drug carriers and the factors controlling their performance. This study aimed to use biodegradable polymer microspheres as alternative carriers to lactose in DPIs for achieving efficient drug delivery into the lungs. This project focused on fabricating biodegradable polymer microparticles with reproducible surface morphology and particle shape. The surface characteristics of polymeric carriers and the adhesional forces between the drug and carrier particles were investigated in order to gain a better understanding of their influence on drug dispersion. For this purpose, two biodegradable polymers- polycaprolactone (PCL) and poly (DL-lactide-co-glycolide) (PLGA) were used as the carriers to deliver the anti-asthmatic drug - Salbutamol Sulphate (SS). The first study conducted for this dissertation was the aerosolization of SS from mixtures of SS and PCL or PLGA microparticles. The microparticles were fabricated using an emulsion technique and were characterized by laser diffraction for particle size analysis, Scanning Electron Microscopy (SEM) for surface morphology and X-ray Photoelectron Spectroscopy (XPS) to obtain surface elemental composition. The dispersion of the drug from the DPI formulations was determined by using a Twin Stage Impinger (TSI). The Fine particle Fraction (FPF) of SS from powder mixtures was analyzed by High Performance Liquid Chromatography (HPLC). It was found that the drug did not detach from the surface of PCL microspheres. To overcome this, the microspheres were coated with anti-adherent agents such as magnesium stearate and leucine to improve the dispersion of the drug from the carrier surfaces. It was found that coating the PCL microspheres helped in significantly improving the FPF of SS from the PCL surface. These results were in contrast to the PLGA microspheres which readily allowed detachment of the SS from their surface. However, coating PLGA microspheres with antiadherent agents did not further improve the detachment of the drug from the surface. Thus, the first part of the study demonstrated that the surface-coated PCL microspheres and PLGA microspheres can be potential alternatives to lactose as carriers in DPI formulations; however, there was no significant improvement in the FPF of the drug. The second part of the research studied the influence of the size of the microspheres on the FPF of the drug. For this purpose, four different sizes (25 ìm, 48 ìm, 100 ìm and 150 ìm) of the PCL and PLGA microspheres were fabricated and characterized. The dispersion of the drug from microspheres of different sizes was determined. It was found that as the size of the carrier increased there was a significant increase in the FPF of SS. This study suggested that the size of the carrier plays an important role in the dispersion of the drug from the carrier surface. Subsequent experiments in the third part of the dissertation studied the surface properties of the polymeric carrier. The adhesion forces existing between the drug particle and the polymer surfaces, and the surface roughness of the carriers were quantified using Atomic Force Microscopy (AFM). A direct correlation between adhesion forces and dispersion of the drug from the carrier surface was observed suggesting that adhesion forces play an important role in determining the detachment potential of the drug from the carrier surface. However, no direct relationship between the surface roughness of the PCL or PLGA carrier and the FPF of the drug was observed. In conclusion, the body of work presented in this dissertation demonstrated the potential of coated PCL microspheres and PLGA microspheres to be used in DPI formulations as an alternative carrier to sugar based carriers. The study also emphasized the role of the size of the carrier particles and the forces of interaction prevailing between the drug and the carrier particle surface on the aerosolization performances of the drug.

Effects of rice husk biochar and sugar-mill by-products on methane consumption from two different soils

Methane (CH4) is an important greenhouse gas with a global warming potential (GWP) 25 times greater than carbon dioxide (CO2) that can be produced or consumed in soils depending on environmental conditions and other factors. Biochar application to soils has been shown to reduce CH4 emissions and to increase CH4 consumption. However, the effects of rice husk biochar (RB) have not been thoroughly investigated. Two 60-day laboratory incubation experiments were conducted to investigate the effects of amending two soil types with RB, raw mill mud (MM) and composted mill mud (CM) on soil CH4 consumption and emissions. Soil cores incubated in 1 L glass jars and gas samples were analysed for CH4 using gas chromatography. Average CH4 consumption rates varied from -0.06 to -0.68 g CH4-C( )/ha/d in sandy loam soil and -0.59 to -1.00 g CH4-C/ha/d in clay soil. Application of RB resulted in CH4 uptake of -0.52 to -0.55 g CH4-C/ha/d in sandy loam and -0.76 to -0.91 g CH4-C/ha/d in clay soil. Addition of MM showed low CH4 emissions or consumption at 60% water-filled pore space (WFPS) in both soils. However, at high water contents (>75% WFPS) the application of MM produced high rates of CH4 emissions which were significantly suppressed when RB was added. Cumulative emissions of the MM treatment produced 108.9 g CH4-C/ha at 75% WFPS and 11 459.3 g CH4-C/ha at 90% WFPS in sandy loam soil over a period of 60 days. RB can increase CH4 uptake under low soil water content (SWC) and decrease CH4 emissions under anaerobic conditions. CM expressed more potential to reduce CH4 emissions than those of MM.

Metabolic and ecological study of environmental pentose utilizing bacteria (E-PUB)

Lignocellulosic materials, such as sugar cane bagasse, a waste product of the sugarcane processing industry, agricultural residues and herbaceous crops, may serve as an abundant and comparatively cheap feedstock for largescale industrial fermentation, resulting in the production of marketable end-products. However, the complex structure of lignocellulosic materials, the presence of various hexose and pentose sugars in the hemicellulose component, and the presence of various compounds that inhibit the organisms selected for the fermentation process, all constitute barriers that add to the production costs and make full scale industrial production economically less feasible. The work presented in this thesis was conducted in order to screen microorganisms for ability to utilize pentose sugars derived from the sugar mill industrial waste. A large number of individual bacterial strains were investigated from hemi-cellulose rich material collected at the Proserpine and Maryborough sugar mills, notably soil samples from the mill sites. The research conducted to isolation of six pentose-capable Gram-positive organisms from the actinomycetes group by using pentose as a sole carbon source in the cultivation process. The isolates were identified as Corynebacterium glutamicum, Actinomyces odontolyticus, Nocardia elegans, and Propionibacterium freudenreichii all of which were isolated from the hemicellulose-enriched soil. Pentose degrading microbes are very rare in the environment, so this was a significant discovery. Previous research indicated that microbes could degrade pentose after genetic modification but the microbes discovered in this research were able to naturally utilize pentose. Six isolates, identified as four different genera, were investigated for their ability to utilize single sugars as substrates (glucose, xylose, arabinose or ribose), and also dual sugars as substrates (a hexose plus a pentose). The results demonstrated that C. glutamicum, A. odontolyticus, N. elegans, and P. freudenreichii were pentose-capable (able to grow using xylose or other pentose sugar), and also showed diauxie growth characteristics during the dual-sugar (glucose, in combination with xylose, arabinose or ribose) carbon source tests. In addition, it was shown that the isolates displayed very small differences in growth rates when grown on dual sugars as compared to single sugars, whether pentose or hexose in nature. The anabolic characteristics of C. glutamicum, A. odontolyticus, N. elegans and P. freudenreichii were subsequently investigated by qualitative analysis of their end-products, using high performance liquid chromatography (HPLC). All of the organisms produced arginine and cysteine after utilization of the pentose substrates alone. In addition, P. freudenreichii produced alanine and glycine. The end-product profile arising from culture with dual carbon sources was also tested. Interestingly, this time the product was different. All of them produced the amino acid glycine, when grown on a combination substrate-mix of glucose with xylose, and also glucose with arabinose. Only N. elegans was able to break down ribose, either singly or in combination with glucose, and the end-product of metabolism of the glucose plus ribose substrate combination was glutamic acid. The ecological analysis of microbial abundance in sugar mill waste was performed using denaturing gradient gel electrophoresis (DGGE) and also the metagenomic microarray PhyloChip method. Eleven solid samples and seven liquid samples were investigated. A very complex bacterial ecosystem was demonstrated in the seven liquid samples after testing with the PhyloChip method. It was also shown that bagasse leachate was the most different, compared to all of the other samples, by virtue of its richness in variety of taxa and the complexity of its bacterial community. The bacterial community in solid samples from Proserpine, Mackay and Maryborough sugar mills showed huge diversity. The information found from 16S rDNA sequencing results was that the bacterial genera Brevibacillus, Rhodospirillaceae, Bacillus, Vibrio and Pseudomonas were present in greatest abundance. In addition, Corynebacterium was also found in the soil samples. The metagenomic studies of the sugar mill samples demonstrate two important outcomes: firstly that the bagasse leachate, as potentially the most pentose-rich sample tested, had the most complex and diverse bacterial community; and secondly that the pentose-capable isolates that were initially discovered at the beginning of this study, were not amongst the most abundant taxonomic groups discovered in the sugar mill samples, and in fact were, as suspected, very rare. As a bioprospecting exercise, therefore, the study has discovered organisms that are naturally present, but in very small numbers, in the appropriate natural environment. This has implications for the industrial application of E-PUB, in that a seeding process using a starter culture will be necessary for industrial purposes, rather than simply assuming that natural fermentation might occur.

Advantages of tandem LC-MS for the rapid assessment of tissue-specific metabolic complexity using a pentafluorophenylpropyl stationary phase

In this study, a tandem LC-MS (Waters Xevo TQ) MRM-based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice, including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a "fingerprint" characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum, and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples.

Metabolomic analysis characterizes tissue specific indomethacin-induced metabolic perturbations of rats

In this study, the promising metabolomic approach integrating with ingenuity pathway analysis (IPA) was applied to characterize the tissue specific metabolic perturbation of rats that was induced by indomethacin. The selective pattern recognition analyses were applied to analyze global metabolic profiling of urine of rats treated by indomethacin at an acute dosage of reference that has been proven to induce tissue disorders in rats, evaluated throughout the time-course of -24-72 h. The results preliminarily revealed that modifications of amino acid metabolism, fatty acid metabolism and energetically associated metabolic pathways accounted for metabolic perturbation of the rats that was induced by indomethacin. Furthermore, IPA was applied to deeply analyze the biomarkers and their relations with the metabolic perturbations evidenced by pattern recognition analyses. Specific biochemical functions affected by indomethacin suggested that there is an important correlation of its effects in kidney and liver metabolism, based on the determined metabolites and their pathway-based analysis. The IPA correlation of the three major biomarkers, identified as creatinine, prostaglandin E2 and guanosine, suggested that the administration of indomethacin induced certain levels of toxicity in the kidneys and liver. The changes in the levels of biomarker metabolites allowed the phenotypical determination of the metabolic perturbations induced by indomethacin in a time-dependent manner.

Development of an integrated metabolomic profiling approach for infectious diseases research

Metabolomic profiling offers direct insights into the chemical environment and metabolic pathway activities at sites of human disease. During infection, this environment may receive important contributions from both host and pathogen. Here we apply an untargeted metabolomics approach to identify compounds associated with an E. coli urinary tract infection population. Correlative and structural data from minimally processed samples were obtained using an optimized LC-MS platform capable of resolving ~2300 molecular features. Principal component analysis readily distinguished patient groups and multiple supervised chemometric analyses resolved robust metabolomic shifts between groups. These analyses revealed nine compounds whose provisional structures suggest candidate infection-associated endocrine, catabolic, and lipid pathways. Several of these metabolite signatures may derive from microbial processing of host metabolites. Overall, this study highlights the ability of metabolomic approaches to directly identify compounds encountered by, and produced from, bacterial pathogens within human hosts.

Thyroxine and reserpine-induced changes in metabolic profiles of rat urine and the therapeutic effect of Liu Wei Di Huang Wan detected by UPLC-HDMS

The promise of metabonomics, a new "omics" technique, to validate Chinese medicines and the compatibility of Chinese formulas has been appreciated. The present study was undertaken to explore the excretion pattern of low molecular mass metabolites in the male Wistar-derived rat model of kidney yin deficiency induced with thyroxine and reserpine as well as the therapeutic effect of Liu Wei Di Huang Wan (LW) and its separated prescriptions, a classic traditional Chinese medicine formula for treating kidney yin deficiency in China. The study utilized ultra-performance liquid chromatography/electrospray ionization synapt high definition mass spectrometry (UPLC/ESI-SYNAPT-HDMS) in both negative and positive electrospray ionization (ESI). At the same time, blood biochemistry was examined to identify specific changes in the kidney yin deficiency. Distinct changes in the pattern of metabolites, as a result of daily administration of thyroxine and reserpine, were observed by UPLC-HDMS combined with a principal component analysis (PCA). The changes in metabolic profiling were restored to their baseline values after treatment with LW according to the PCA score plots. Altogether, the current metabonomic approach based on UPLC-HDMS and orthogonal projection to latent structures discriminate analysis (OPLS-DA) indicated 20 ions (14 in the negative mode, 8 in the positive mode, and 2 in both) as "differentiating metabolites".

Ingenuity pathways analysis of urine metabonomics phenotypes toxicity of gentamicin in multiple organs

We introduce the use of Ingenuity Pathway Analysis to analyzing global metabonomics in order to characterize phenotypically biochemical perturbations and the potential mechanisms of the gentamicin-induced toxicity in multiple organs. A single dose of gentamicin was administered to Sprague Dawley rats (200 mg/kg, n = 6) and urine samples were collected at -24-0 h pre-dosage, 0-24, 24-48, 48-72 and 72-96 h post-dosage of gentamicin. The urine metabonomics analysis was performed by UPLC/MS, and the mass spectra signals of the detected metabolites were systematically deconvoluted and analyzed by pattern recognition analyses (Heatmap, PCA and PLS-DA), revealing a time-dependency of the biochemical perturbations induced by gentamicin toxicity. As result, the holistic metabolome change induced by gentamicin toxicity in the animal's organisms was characterized. Several metabolites involved in amino acid metabolism were identified in urine, and it was confirmed that gentamicin biochemical perturbations can be foreseen from these biomarkers. Notoriously, it was found that gentamicin induced toxicity in multiple organs system in the laboratory rats. The proof-of-knowledge based Ingenuity Pathway Analysis revealed gentamicin induced liver and heart toxicity, along with the previously known toxicity in kidney. The metabolites creatine, nicotinic acid, prostaglandin E2, and cholic acid were identified and validated as phenotypic biomarkers of gentamicin induced toxicity. Altogether, the significance of the use of metabonomics analyses in the assessment of drug toxicity is highlighted once more; furthermore, this work demonstrated the powerful predictive potential of the Ingenuity Pathway Analysis to study of drug toxicity and its valuable complementation for metabonomics based assessment of the drug toxicity.

Plasma metabolomics reveals biomarkers of the atherosclerosis

Atherosclerotic cardiovascular disease remains the leading cause of morbidity and mortality in industrialized societies. The lack of metabolite biomarkers has impeded the clinical diagnosis of atherosclerosis so far. In this study, stable atherosclerosis patients (n=16) and age- and sex-matched non-atherosclerosis healthy subjects (n=28) were recruited from the local community (Harbin, P. R. China). The plasma was collected from each study subject and was subjected to metabolomics analysis by GC/MS. Pattern recognition analyses (principal components analysis, orthogonal partial least-squares discriminate analysis, and hierarchical clustering analysis) commonly demonstrated plasma metabolome, which was significantly different from atherosclerotic and non-atherosclerotic subjects. The development of atherosclerosis-induced metabolic perturbations of fatty acids, such as palmitate, stearate, and 1-monolinoleoylglycerol, was confirmed consistent with previous publication, showing that palmitate significantly contributes to atherosclerosis development via targeting apoptosis and inflammation pathways. Altogether, this study demonstrated that the development of atherosclerosis directly perturbed fatty acid metabolism, especially that of palmitate, which was confirmed as a phenotypic biomarker for clinical diagnosis of atherosclerosis.

Simultaneous determination by UPLC-ESI-MS of scoparone, capillarisin, rhein, and emodin in rat urine after oral administration of Yin Chen Hao Tang preparation

A simple, sensitive, and validated method was developed for simultaneous determination of scoparone, capillarisin, rhein, and emodin in rat urine by ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (UPLC-MS). The urinary samples were analyzed on an Acquity UPLC BEH C18 1.7 microm 2.1x50 mm column. Scoparone, capillarisin, rhein, and emodin in rat urine were simultaneously analyzed with good separation. The lower limits of detection were 6.0, 9.0, 7.0, and 3.0 ng/mL, and the lower limits of quantification were 20.0, 33.0, 24.0, and 12.0 ng/mL for scoparone, capillarisin, rhein, and emodin, respectively. The intra- and inter-day precisions (RSD) were less than 9%. The intra- and inter-accuracies were found to be in the range of 94.14-104.54% for scoparone, 101.72-107.34% for capillarisin, 95.24-103.59% for rhein, and 101.32-107.82% for emodin at three concentration levels. The absolute recoveries for scoparone, capillarisin, rhein, and emodin were not less than 77.0%. The developed method has been applied to determine scoparone, capillarisin, rhein, and emodin in rat urine after oral administration of Yin Chen Hao Tang preparation, a traditional Chinese medicine formulation widely used in China for treatment of jaundice and liver disorders.