Publications by authors named "Dirk K Wissenbach"

45 Publications

HPLC-MS identification of acid degradation products of dolutegravir.

J Pharm Biomed Anal 2021 Feb 6;197:113954. Epub 2021 Feb 6.

Friedrich Schiller University, Department of Pharmaceutical/Medicinal Chemistry, Philosophenweg 14, D-07743 Jena, Germany. Electronic address:

Dolutegravir is an integrase strand transfer inhibitor used for the treatment of human immuno-deficiency virus infections. The present study was conducted in order to identify degradation products formed in acidic solution upon heating. The structures were assigned based on low resolution collision-induced dissociation tandem mass spectra as well as high resolution higher-energy collisional dissociation tandem mass spectra. The major degradation products resulted from hydrolytic opening of the oxepine ring leading to bis-hydroxy diastereomers (DP2 and DP3) as well as a mono-hydroxy derivative (DP1) as the result of dehydration of the diastereomers. Furthermore, two carboxylic acid derivatives (DP4 and DP5) could be identified, which can be explained as the result of the hydrolysis of the exocyclic amide bond of dolutegravir and DP1, respectively. During the fragmentation process of dolutegravir and its degradation products DP1 to DP3 a formal addition of oxygen resulting in the respective carboxylic acid fragments was detected. This could be evidenced based on high resolution masses of the fragments as well as the comparison of the MS/MS spectra of the fragments with the spectra of the carboxylic acids DP4 and DP5.
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http://dx.doi.org/10.1016/j.jpba.2021.113954DOI Listing
February 2021

Surviving chlormequat poisoning - pharmacokinetics and the role of atropine.

Clin Toxicol (Phila) 2021 Jan 27;59(1):74-76. Epub 2020 Apr 27.

Division of Clinical Toxicology and Poison Control Centre Munich, Department of Internal Medicine II, School of Medicine, Technical University of Munich, Munich, Germany.

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http://dx.doi.org/10.1080/15563650.2020.1758326DOI Listing
January 2021

Ethnomedicinal survey and in vitro confirmation of anti-inflammatory and antispasmodic properties of the termite strain Macrotermes bellicosus used in traditional medicine in the Republic of Benin.

J Ethnopharmacol 2020 May 22;254:112705. Epub 2020 Feb 22.

Leipzig University, Medical Faculty, Institute for Medical Physics and Biophysics, Leipzig, Germany. Electronic address:

Ethnopharmacological Relevance: Insects and insect-derived products play a vital role in traditional medicine in many parts of the world since ancient times. Among these insects, fungus-growing termites like Macrotermes bellicosus (M. bellicosus) are widely used in nutrition and traditional medicine in various societies of sub-Saharan Africa.

Aim Of The Study: Aim of the present study was to explore the traditional applications of M. bellicosus and subsequently investigate the anti-inflammatory and spasmolytic activity of samples collected in Benin.

Material And Methods: An ethnomedicinal survey with thirty active healers in Benin was conducted and the anti-inflammatory activity of an ethanolic extract of M. bellicosus was investigated. Thus, LPS-induced TNFα release from differentiated human macrophages (THP-1) and IL-8 release from cytokine (IL-1β/TNFα/IFNγ)-challenged human intestinal epithelial cells (Caco-2) was measured by enzyme-linked immunosorbent assay. Furthermore, the influence of M. bellicosus extract on basal tone and induced contractions in isolated rat small intestinal preparations was determined to examine the influence on intestinal motility.

Results: The survey of 30 active healers demonstrated that M. bellicosus and its products (termites' mound and fungus comb) are used in Benin for therapeutic purposes mainly to treat infectious and inflammatory diseases including digestive disorders, snake bites and diarrhea. It was found that M. bellicosus extract inhibited both LPS-induced TNFα release from human macrophages and cytokine-induced IL-8 release from intestinal epithelial cells comparable to budesonide. In addition, isometric contraction measurement with isolated rat small intestinal preparations demonstrated a mild spasmolytic effect of the termite extract in higher concentrations with a suppression of induced contractions and relaxation of basal tone.

Conclusion: M. bellicosus which is used in traditional medicine in Benin to treat infectious and inflammatory diseases showed anti-inflammatory activity by inhibiting pro-inflammatory cytokine release and a moderate influence on intestinal motility.
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http://dx.doi.org/10.1016/j.jep.2020.112705DOI Listing
May 2020

Gastric bypass surgery in a rat model alters the community structure and functional composition of the intestinal microbiota independently of weight loss.

Microbiome 2020 02 7;8(1):13. Epub 2020 Feb 7.

Department of Molecular Systems Biology, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany.

Background: Roux-en-Y gastric bypass (RYGB) surgery is a last-resort treatment to induce substantial and sustained weight loss in cases of severe obesity. This anatomical rearrangement affects the intestinal microbiota, but so far, little information is available on how it interferes with microbial functionality and microbial-host interactions independently of weight loss.

Methods: A rat model was employed where the RYGB-surgery cohort is compared to sham-operated controls which were kept at a matched body weight by food restriction. We investigated the microbial taxonomy and functional activity using 16S rRNA amplicon gene sequencing, metaproteomics, and metabolomics on samples collected from theileum, the cecum, and the colon, and separately analysed the lumen and mucus-associated microbiota.

Results: Altered gut architecture in RYGB increased the relative occurrence of Actinobacteria, especially Bifidobacteriaceae and Proteobacteria, while in general, Firmicutes were decreased although Streptococcaceae and Clostridium perfringens were observed at relative higher abundances independent of weight loss. A decrease of conjugated and secondary bile acids was observed in the RYGB-gut lumen. The arginine biosynthesis pathway in the microbiota was altered, as indicated by the changes in the abundance of upstream metabolites and enzymes, resulting in lower levels of arginine and higher levels of aspartate in the colon after RYGB.

Conclusion: The anatomical rearrangement in RYGB affects microbiota composition and functionality as well as changes in amino acid and bile acid metabolism independently of weight loss. The shift in the taxonomic structure of the microbiota after RYGB may be mediated by the resulting change in the composition of the bile acid pool in the gut and by changes in the composition of nutrients in the gut. Video abstract.
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http://dx.doi.org/10.1186/s40168-020-0788-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007695PMC
February 2020

Liquid chromatography-mass spectrometry-based determination of ergocristine, ergocryptine, ergotamine, ergovaline, hypoglycin A, lolitrem B, methylene cyclopropyl acetic acid carnitine, N-acetylloline, N-formylloline, paxilline, and peramine in equine hair.

J Chromatogr B Analyt Technol Biomed Life Sci 2019 Jun 5;1117:127-135. Epub 2019 Apr 5.

Institute of Forensic Medicine, Jena University Hospital, Jena, Germany. Electronic address:

Ingestion of hypoglycin A (HGA) in maple seeds or alkaloids produced by symbiotic fungi in pasture grasses is thought to be associated with various syndromes in grazing animals. This article describes analytical methods for monitoring long-term exposure to HGA, its metabolite MCPA-carnitine, as well as ergocristine, ergocryptine, ergotamine, ergovaline, lolitrem B, N-acetylloline, N-formylloline, peramine, and paxilline in equine hair. After extraction of hair samples separation was achieved using two ultra high performance liquid chromatographic systems (HILIC or RP-C18, ammonium formate:acetonitrile). A benchtop orbitrap instrument was used for high resolution tandem mass spectrometric detection. All analytes were sensitively detected with limits of detection between 1 pg/mg and 25 pg/mg. Irreproducible extraction or ubiquitous presence in horse hair precluded quantitative validation of lolitrem B/paxilline and N-acetylloline/N-formylloline, respectively. For the other analytes validation showed no interferences in blank hair. Other validation parameters were as follows: limits of quantification (LOQ), 10 to 100 pg/mg; recoveries, 18.3 to 91.0%; matrix effects, -48.2 - 24.4%; linearity, LOQ - 1000 pg/mg; accuracy, -14.9 - 6.4%, precision RSDs ≤10.7%. The method allows sensitive detection of all analytes and quantification of ergocristine, ergocryptine, ergotamine, ergovaline, HGA, MCPA-carnitine, and peramine in horse hair. Applicability was proven for N-acetylloline and N-formylloline by analyzing hair of 13 horses.
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http://dx.doi.org/10.1016/j.jchromb.2019.04.016DOI Listing
June 2019

Comparative study on the metabolism of the ergot alkaloids ergocristine, ergocryptine, ergotamine, and ergovaline in equine and human S9 fractions and equine liver preparations.

Xenobiotica 2019 Oct 9;49(10):1149-1157. Epub 2019 Jan 9.

a Institute of Forensic Medicine , Jena University Hospital , Jena , Germany.

1. Ergopeptine alkaloids like ergovaline and ergotamine are suspected to be associated with fescue toxicosis and ergotism in horses. Information on the metabolism of ergot alkaloids is scarce, especially in horses, but needed for toxicological analysis of these drugs in urine/feces of affected horses. The aim of this study was to investigate the metabolism of ergovaline, ergotamine, ergocristine, and ergocryptine in horses and comparison to humans. 2. Supernatants of alkaloid incubations with equine and human liver S9 fractions were analyzed by reversed-phase liquid-chromatography coupled to high-resolution tandem mass spectrometry with full scan and MS acquisition. Metabolite structures were postulated based on their MS spectra in comparison to those of the parent alkaloids. All compounds were extensively metabolized yielding nor-, -oxide, hydroxy and dihydro-diole metabolites with largely overlapping patterns in equine and human liver S9 fractions. However, some metabolic steps e.g. the formation of 8'-hydroxy metabolites were unique for human metabolism, while formation of the 13/14-hydroxy and 13,14-dihydro-diol metabolites were unique for equine metabolism. Incubations with equine whole liver preparations yielded less metabolites than the S9 fractions. 3. The acquired data can be used to develop metabolite-based screenings for these alkaloids, which will likely extend their detection windows in urine/feces from affected horses.
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http://dx.doi.org/10.1080/00498254.2018.1542187DOI Listing
October 2019

Systematic investigations of novel validity parameters in urine drug testing and prevalence of urine adulteration in a two-year cohort.

Drug Test Anal 2018 Oct 7;10(10):1536-1542. Epub 2018 Aug 7.

Institute of Forensic Medicine, Jena University Hospital, Jena, Germany.

Urinalysis is well established for drug screening. Various methods of urine adulteration such as dilution, addition of oxidative/reductive chemicals or detergents, and handing over urine-like fluids are used to circumvent a positive screen. Validity parameters such as determination of pH, gravidity, urine temperature, or testing for oxidative/reductive chemicals are therefore used to uncover adulterated urine specimens. However, synthetic urine ("fake urine") has nowadays been used for manipulations, leading to inconspicuous results with common validity test systems. Therefore, the aims of the study were (a) to evaluate additional validity parameters, (b) to evaluate the prevalence of urine adulteration, (c) to identify adulteration markers in purchased fake urine samples. Urine samples (n = 550) submitted for drug abstinence testing were analyzed by a standard urine liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening approach using library-assisted identification of 10 different endogenous biomolecules. The detection rates of biomolecules in authentic samples were phenylalanine (93.4%), tryptophan (97.1%), propionyl-carnitine (67.1%), butyryl-carnitine (99.6%), isovaleryl-carnitine (92.8%), hexanoyl-carnitine (91.0%), heptanoyl-carnitine (97.1%), octanoyl-carnitine (98.9%), and indoleacetylglutamine (98,2%). Phenylacetylglutamine was detected in each authentic sample. Based on the detection rates and measured creatinine levels, six manipulated samples were identified in this study. In two cases, fake urine was handed over, one time fake urine was most likely used for dilution. Once dilution with other fluids was used as adulteration method, while in another sample a detergent solution was handed over. Additionaly, one sample contained reactive chemicals. All fake urine samples were additionally identified by the detection of unique polyglycole patterns, which were observed in purchased fake urine samples.
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http://dx.doi.org/10.1002/dta.2447DOI Listing
October 2018

Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry assay for nine toxic alkaloids from endophyte-infected pasture grasses in horse serum.

J Chromatogr A 2018 Jul 7;1560:35-44. Epub 2018 May 7.

Institute of Forensic Medicine, Jena University Hospital, Jena, Germany. Electronic address:

Endophyte fungi (e.g. Epichloë ssp. and Neotyphodium ssp.) in symbiosis with pasture grasses (e.g. Festuca arundinacaea and Lolium perenne) can produce toxic alkaloids, which are suspected to be involved in equine diseases such as fescue toxicosis, ryegrass staggers, and equine fescue oedema. The aim of this study was, therefore, to develop and validate a quantification method for these and related alkaloids: ergocristine, ergocryptine, ergotamine, ergovaline, lolitrem B, lysergic acid, N-acetylloline, N-formylloline, peramine, and paxilline in horse serum. Horse serum samples (1.5mL) were worked up by solid-phase extraction (OASIS HLB). The extracts were analyzed by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS). Chromatographic separation was achieved by gradient elution with ammonium formate buffer and acetonitrile on a RP18 column (100×2.1mm; 1.7μm). HRMS/MS detection was performed on a QExactive Focus instrument with heated positive electrospray ionization and operated in the parallel reaction monitoring (PRM) mode. Method validation included evaluation of selectivity, matrix effect, recovery, linearity, limit of quantification (LOQ), limit of detection (LOD), accuracy, and stability. With exception of lolitrem B solid phase extraction yielded high recoveries (73.6-104.6%) for all analytes. Chromatographic separation of all analytes was achieved with a run time of 25min. HRMS/MS allowed sensitive detection with LODs ranging from 0.05 to 0.5ng/mL and LOQs from 0.1 to 1.0ng/mL. Selectivity experiments showed no interferences from matrix or IS, but N-acetylloline and N-formylloline were found to be ubiquitous in horse serum. Newborn calf serum was therefore used as surrogate matrix for the validation study. Calibration ranges were analyte-dependent and in total covered concentrations from 0.1 to 50ng/mL. Lolitrem B and paxilline could be sensitively detected, but did not meet quantification requirements. For the other analytes, accuracy and precision were shown for 3 different concentrations (QC low, medium, high) with acceptable bias (-10, 5%-7.9%) and precision (CV 2.6%-12.5%). Matrix effects varied from 55.0% to 121% (RSD 7.8-18.5%) and were adequately compensated by IS. Matrix effects of N-acetylloline and N-formylloline could only be estimated in newborn calf serum (n=1) and ranged from 52.5-88.3%. All analytes were stable under autosampler conditions and over 3 freeze and thaw cycles. Applicability was proven by analyzing authentic horse serum samples (n=24). In conclusion, the presented method allows a sensitive detection of ergocrisitine, ergocryptine, ergotamine, ergovaline, lolitrem B, lysergic acid, N-acetylloline, N-formylloline, peramine, and paxilline in horse serum and reliable quantification of all but lolitrem B and paxilline.
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http://dx.doi.org/10.1016/j.chroma.2018.05.013DOI Listing
July 2018

mediates the impact of prenatal bisphenol A exposure on long-term body weight development.

Clin Epigenetics 2018 20;10:58. Epub 2018 Apr 20.

1Department of Environmental Immunology, Helmholtz Centre for Environmental Research (UFZ), Leipzig, Germany.

Background: Exposure to endocrine-disrupting chemicals can alter normal physiology and increase susceptibility to non-communicable diseases like obesity. Especially the prenatal and early postnatal period is highly vulnerable to adverse effects by environmental exposure, promoting developmental reprogramming by epigenetic alterations. To obtain a deeper insight into the role of prenatal bisphenol A (BPA) exposure in children's overweight development, we combine epidemiological data with experimental models and BPA-dependent DNA methylation changes.

Methods: BPA concentrations were measured in maternal urine samples of the LINA mother-child-study obtained during pregnancy ( = 552), and BPA-associated changes in cord blood DNA methylation were analyzed by Illumina Infinium HumanMethylation450 BeadChip arrays ( = 472). Methylation changes were verified by targeted MassARRAY analyses, assessed for their functional translation by qPCR and correlated with children's body mass index (BMI) scores at the age of 1 and 6 years. Further, female BALB/c mice were exposed to BPA from 1 week before mating until delivery, and weight development of their pups was monitored ( ≥ 8/group). Additionally, human adipose-derived mesenchymal stem cells were treated with BPA during the adipocyte differentiation period and assessed for exposure-related epigenetic, transcriptional and morphological changes ( = 4).

Results: In prenatally BPA-exposed children two CpG sites with deviating cord blood DNA-methylation profiles were identified, among them a hypo-methylated CpG in the promoter of the obesity-associated mesoderm-specific transcript (. A mediator analysis suggested that prenatal BPA exposure was connected to cord blood promoter methylation and expression as well as BMI scores in early infancy. This effect could be confirmed in mice in which prenatal BPA exposure altered promoter methylation and transcription with a concomitant increase in the body weight of the juvenile offspring. An experimental model of in vitro differentiated human mesenchymal stem cells also revealed an epigenetically induced expression and enhanced adipogenesis following BPA exposure.

Conclusions: Our study provides evidence that mediates the impact of prenatal BPA exposure on long-term body weight development in offspring by triggering adipocyte differentiation.
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http://dx.doi.org/10.1186/s13148-018-0478-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910578PMC
May 2019

Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry quantification method for hypoglycin A and methylene cyclopropyl acetic acid carnitine in horse serum in cases of atypical myopathy.

Drug Test Anal 2018 May 12;10(5):814-820. Epub 2017 Dec 12.

Institute of Forensic Medicine, Jena University Hospital, Jena, Germany.

Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycine A (HGA) from ingested maple seeds and its active metabolite methylene cyclopropyl acetic acid (MCPA). The aim of this study was the development and validation of a rapid and simple assay for HGA and MCPA-carnitine in horse serum and its application to authentic samples. Identification and quantification were carried out by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with full-scan/data-dependent MS/MS. Chromatographic separation was performed by isocratic elution on a hydrophilic interaction liquid chromatography (HILIC) column (100 x 2.1 mm, 1.7 μm). Serum samples (250 μL) were worked up by protein precipitation. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, matrix effects, and recovery. The calibration range was from 100 to 2000 ng/mL for HGA and from 10 to 1000 ng/mL for MCPA-carnitine. HGA and MCPA-carnitine showed acceptable accuracy and precision (bias -3.0% to 1.1%; RSD 9.2% to 12.4%). The limit of quantification (LOQ) was defined as the lowest calibrator and well below the lowest published serum concentrations in affected horses. Matrix effects ranged from -79% to +20% (RSD 4.2% to 14.4%), recoveries from 17.9% to 21.1% (RSD 2.3% to 10.8 %) for low and high quality control samples, respectively. Applicability was tested in 10 authentic AM cases. In all specimens, relevant amounts of HGA and MCPA-carnitine were found (570-2000 ng/mL; ~8.5-150 ng/mL, respectively). The developed assay allows reliable identification and quantification of HGA and MCPA-carnitine in horse serum and will be helpful to further study the association between HGA/MCPA and AM.
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http://dx.doi.org/10.1002/dta.2337DOI Listing
May 2018

Acidotolerant and Fungi as a Sink of Methanol-Derived Carbon in a Deciduous Forest Soil.

Front Microbiol 2017 24;8:1361. Epub 2017 Jul 24.

Department of Ecological Microbiology, University of BayreuthBayreuth, Germany.

Methanol is an abundant atmospheric volatile organic compound that is released from both living and decaying plant material. In forest and other aerated soils, methanol can be consumed by methanol-utilizing microorganisms that constitute a known terrestrial sink. However, the environmental factors that drive the biodiversity of such methanol-utilizers have been hardly resolved. Soil-derived isolates of methanol-utilizers can also often assimilate multicarbon compounds as alternative substrates. Here, we conducted a comparative DNA stable isotope probing experiment under methylotrophic (only [C]-methanol was supplemented) and combined substrate conditions ([C]-methanol and alternative multi-carbon [C]-substrates were simultaneously supplemented) to (i) identify methanol-utilizing microorganisms of a deciduous forest soil (European beech dominated temperate forest in Germany), (ii) assess their substrate range in the soil environment, and (iii) evaluate their trophic links to other soil microorganisms. The applied multi-carbon substrates represented typical intermediates of organic matter degradation, such as acetate, plant-derived sugars (xylose and glucose), and a lignin-derived aromatic compound (vanillic acid). An experimentally induced pH shift was associated with substantial changes of the diversity of active methanol-utilizers suggesting that soil pH was a niche-defining factor of these microorganisms. The main bacterial methanol-utilizers were members of the () that played a central role in a detected methanol-based food web. A clear preference for methanol or multi-carbon substrates as carbon source of different -affiliated phylotypes was observed suggesting a restricted substrate range of the methylotrophic representatives. Apart from , we also identified the yeasts and as methanol-derived carbon-utilizing fungi suggesting that further research is needed to exclude or prove methylotrophy of these fungi.
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http://dx.doi.org/10.3389/fmicb.2017.01361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5523551PMC
July 2017

Method Development in Forensic Toxicology.

Curr Pharm Des 2017 ;23(36):5455-5467

National Centre On Addiction And Doping, Istituto Superiore di Sanita, Rome, Italy.

Background: In the field of forensic toxicology, the quality of analytical methods is of great importance to ensure the reliability of results and to avoid unjustified legal consequences. A key to high quality analytical methods is a thorough method development.

Methods: The presented article will provide an overview on the process of developing methods for forensic applications.

Results: This includes the definition of the method's purpose (e.g. qualitative vs quantitative) and the analytes to be included, choosing an appropriate sample matrix, setting up separation and detection systems as well as establishing a versatile sample preparation.

Conclusion: Method development is concluded by an optimization process after which the new method is subject to method validation.
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http://dx.doi.org/10.2174/1381612823666170622113331DOI Listing
June 2019

Degradation of indoor limonene by outdoor ozone: A cascade of secondary organic aerosols.

Environ Pollut 2017 Jul 27;226:463-472. Epub 2017 Apr 27.

Department Urban and Environmental Sociology, UFZ Helmholtz Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany. Electronic address:

In indoor air, terpene-ozone reactions can form secondary organic aerosols (SOA) in a transient process. 'Real world' measurements conducted in a furnished room without air conditioning were modelled involving the indoor background of airborne particulate matter, outdoor ozone infiltrated by natural ventilation, repeated transient limonene evaporations, and different subsequent ventilation regimes. For the given setup, we disentangled the development of nucleated, coagulated, and condensed SOA fractions in the indoor air and calculated the time dependence of the aerosol mass fraction (AMF) by means of a process model. The AMF varied significantly between 0.3 and 5.0 and was influenced by the ozone limonene ratio and the background particles which existed prior to SOA formation. Both influencing factors determine whether nucleation or adsorption processes are preferred; condensation is strongly intensified by particulate background. The results provide evidence that SOA levels in natural indoor environments can surpass those known from chamber measurements. An indicator for the SOA forming potential of limonene was found to be limona ketone. Multiplying its concentration (in μg/m) by 450(±100) provides an estimate of the concentration of the reacted limonene. This can be used to detect a high particle formation potential due to limonene pollution, e.g. in epidemiological studies considering adverse health effects of indoor air pollutants.
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http://dx.doi.org/10.1016/j.envpol.2017.04.030DOI Listing
July 2017

Recent advances of liquid chromatography-(tandem) mass spectrometry in clinical and forensic toxicology - An update.

Clin Biochem 2016 Sep 22;49(13-14):1051-71. Epub 2016 Jul 22.

University Hospital Jena, Institute of Forensic Medicine, Fürstengraben 23, D-07743 Jena, Germany. Electronic address:

Liquid chromatography (LC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) is a well-established and widely used technique in clinical and forensic toxicology as well as doping control especially for quantitative analysis. In recent years, many applications for so-called multi-target screening and/or quantification of drugs, poisons, and or their metabolites in biological matrices have been developed. Such methods have proven particularly useful for analysis of so-called new psychoactive substances that have appeared on recreational drug markets throughout the world. Moreover, the evolvement of high resolution MS techniques and the development of data-independent detection modes have opened new possibilities for applications of LC-(MS/MS) in systematic toxicological screening analysis in the so called general unknown setting. The present paper will provide an overview and discuss these recent developments focusing on the literature published after 2010.
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http://dx.doi.org/10.1016/j.clinbiochem.2016.07.010DOI Listing
September 2016

Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles.

PLoS One 2016 21;11(7):e0159580. Epub 2016 Jul 21.

University Center of Orthopedics and Trauma Surgery and Center for Translational Bone, Joint and Soft Tissue Research, University Hospital "Carl Gustav Carus", TU Dresden, Dresden, Germany.

Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0159580PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956113PMC
July 2017

Prenatal maternal stress and wheeze in children: novel insights into epigenetic regulation.

Sci Rep 2016 06 28;6:28616. Epub 2016 Jun 28.

Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg, 69120, Germany.

Psychological stress during pregnancy increases the risk of childhood wheeze and asthma. However, the transmitting mechanisms remain largely unknown. Since epigenetic alterations have emerged as a link between perturbations in the prenatal environment and an increased disease risk we used whole genome bisulfite sequencing (WGBS) to analyze changes in DNA methylation in mothers and their children related to prenatal psychosocial stress and assessed its role in the development of wheeze in the child. We evaluated genomic regions altered in their methylation level due to maternal stress based of WGBS data of 10 mother-child-pairs. These data were complemented by longitudinal targeted methylation and transcriptional analyses in children from our prospective mother-child cohort LINA for whom maternal stress and wheezing information was available (n = 443). High maternal stress was associated with an increased risk for persistent wheezing in the child until the age of 5. Both mothers and children showed genome-wide alterations in DNA-methylation specifically in enhancer elements. Deregulated neuroendocrine and neurotransmitter receptor interactions were observed in stressed mothers and their children. In children but not in mothers, calcium- and Wnt-signaling required for lung maturation in the prenatal period were epigenetically deregulated and could be linked with wheezing later in children's life.
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http://dx.doi.org/10.1038/srep28616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4923849PMC
June 2016

Optimization of metabolomics of defined in vitro gut microbial ecosystems.

Int J Med Microbiol 2016 Aug 16;306(5):280-289. Epub 2016 Mar 16.

Department of Molecular Systems Biology, Helmholtz-Centre for Environmental Research-UFZ, Permoserstrasse 15, D-04318 Leipzig, Germany; Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany; Aalborg University, Department of Chemistry and Biosciences, Aalborg University, 9000 Aalborg, Denmark. Electronic address:

The metabolic functionality of a microbial community is a key to the understanding of its inherent ecological processes and the interaction with the host. However, the study of the human gut microbiota is hindered by the complexity of this ecosystem. One way to resolve this issue is to derive defined communities that may be cultured ex vivo in bioreactor systems and used to approximate the native ecosystem. Doing so has the advantage of experimental reproducibility and ease of sampling, and furthermore, in-depth analysis of metabolic processes becomes highly accessible. Here, we review the use of bioreactor systems for ex vivo modelling of the human gut microbiota with respect to analysis of the metabolic output of the microbial ecosystem, and discuss the possibility of mechanistic insights using these combined techniques. We summarize the different platforms currently used for metabolomics and suitable for analysis of gut microbiota samples from a bioreactor system. With the help of representative datasets obtained from a series of bioreactor runs, we compare the outputs of both NMR and mass spectrometry based approaches in terms of their coverage, sensitivity and quantification. We also discuss the use of untargeted and targeted analyses in mass spectroscopy and how these techniques can be combined for optimal biological interpretation. Potential solutions for linking metabolomic and phylogenetic datasets with regards to active, key species within the ecosystem will be presented.
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http://dx.doi.org/10.1016/j.ijmm.2016.03.007DOI Listing
August 2016

Characterization of chemical-induced sterile inflammation in vitro: application of the model compound ketoconazole in a human hepatic co-culture system.

Arch Toxicol 2017 Feb 10;91(2):799-810. Epub 2016 Mar 10.

Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589, Berlin, Germany.

Liver injury as a result of a sterile inflammation is closely linked to the activation of immune cells, including macrophages, by damaged hepatocytes. This interaction between immune cells and hepatocytes is as yet not considered in any of the in vitro test systems applied during the generation of new drugs. Here, we established and characterized a novel in vitro co-culture model with two human cell lines, HepG2 and differentiated THP-1. Ketoconazole, an antifungal drug known for its hepatotoxicity, was used as a model compound in the testing of the co-culture. Single cultures of HepG2 and THP-1 cells were studied as controls. Different metabolism patterns of ketoconazole were observed for the single and co-culture incubations as well as for the different cell types. The main metabolite N-deacetyl ketoconazole was found in cell pellets, but not in supernatants of cell cultures. Global proteome analysis showed that the NRF2-mediated stress response and the CXCL8 (IL-8) pathway were induced by ketoconazole treatment under co-culture conditions. The upregulation and ketoconazole-induced secretion of several pro-inflammatory cytokines, including CXCL8, TNF-α and CCL3, was observed in the co-culture system only, but not in single cell cultures. Taking together, we provide evidence that the co-culture model applied might be suitable to serve as tool for the prediction of chemical-induced sterile inflammation in liver tissue in vivo.
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http://dx.doi.org/10.1007/s00204-016-1686-yDOI Listing
February 2017

Assessment of maternal drug intake by urinary bio monitoring during pregnancy and postpartally until the third perinatal year.

Pharmacoepidemiol Drug Saf 2016 Apr 22;25(4):431-7. Epub 2015 Dec 22.

Children's Hospital, Municipal Hospital St. Georg Leipzig, affiliated to the University of Leipzig, Leipzig, Germany.

Purpose: Although sales of prescribed and over-the-counter (OTC) medication are rising, little is known about individual drug intake. This study was aimed to obtain complementary information about drug intake.

Method: Information on drug utilization was obtained in a female cohort for five different time points (TP): 36th week of pregnancy (n = 622), 7th perinatal week (n = 533), 3rd perinatal month (n = 340), and 1st perinatal (n = 534) and 3rd perinatal year (n = 324) by a validated urine screening method.

Results: Drugs were detected 807 times among all analyzed samples (n = 2353) with less drug intake for early TP compared with later TP (~24.4%, n = 152; ~33.8%, n = 180; ~23.2%, n = 79; ~42.5%, n = 227; and ~52.2%, n = 169). The diversity of drugs increased from 25 up to 40 different drugs for the investigated period. OTC drugs were detected most frequently reflected by the top three drugs: acetaminophen (~37%, n = 292), ibuprofen (~23%, n = 183), and xylometazoline (~12%, n = 98). Mainly guideline-orientated drug therapy was observed. However, contraindicated ibuprofen intake during third trimester urine samples (n = 26) and a repeated usage of acetaminophen and/or ibuprofen (n = 9), as well as xylometazoline (n = 7), reveal missing information about drug safety.

Conclusion: Bio monitoring was applied for detection of drug intake revealing a lack of information about OTC products and their health risks. Hence, information about health risks for certain drugs and patient groups must be improved for and by pharmacists, to avoid (i) usage of contraindicated drugs and (ii) abuse of OTC drugs.
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http://dx.doi.org/10.1002/pds.3943DOI Listing
April 2016

Genomic, Proteomic, and Metabolite Characterization of Gemfibrozil-Degrading Organism Bacillus sp. GeD10.

Environ Sci Technol 2016 Jan 31;50(2):744-55. Epub 2015 Dec 31.

Aalborg University , Department of Chemistry and Bioscience; Fredrik Bajers Vej 7H, DK-9220 Aalborg, Denmark.

Gemfibrozil is a widely used hypolipidemic and triglyceride lowering drug. Excess of the drug is excreted and discharged into the environment primarily via wastewater treatment plant effluents. Bacillus sp. GeD10, a gemfibrozil-degrader, was previously isolated from activated sludge. It is the first identified bacterium capable of degrading gemfibrozil. Gemfibrozil degradation by Bacillus sp. GeD10 was here studied through genome sequencing, quantitative proteomics and metabolite analysis. From the bacterial proteome of Bacillus sp. GeD10 1974 proteins were quantified, of which 284 proteins were found to be overabundant by more than 2-fold (FDR corrected p-value ≤0.032, fold change (log2) ≥ 1) in response to gemfibrozil exposure. Metabolomic analysis identified two hydroxylated intermediates as well as a glucuronidated hydroxyl-metabolite of gemfibrozil. Overall, gemfibrozil exposure in Bacillus sp. GeD10 increased the abundance of several enzymes potentially involved in gemfibrozil degradation as well as resulted in the production of several gemfibrozil metabolites. The potential catabolic pathway/modification included ring-hydroxylation preparing the substrate for subsequent ring cleavage by a meta-cleaving enzyme. The identified genes may allow for monitoring of potential gemfibrozil-degrading organisms in situ and increase the understanding of microbial processing of trace level contaminants. This study represents the first omics study on a gemfibrozil-degrading bacterium.
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http://dx.doi.org/10.1021/acs.est.5b05003DOI Listing
January 2016

Di-(2-Ethylhexyl)-Phthalate (DEHP) Causes Impaired Adipocyte Function and Alters Serum Metabolites.

PLoS One 2015 2;10(12):e0143190. Epub 2015 Dec 2.

IFB AdiposityDiseases, University of Leipzig, Leipzig, Germany.

Di-(2-ethylhexyl)-phthalate (DEHP), an ubiquitous environmental contaminant, has been shown to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies, but the underlying mechanisms are still unknown. We therefore tested the hypothesis that chronic DEHP exposure causes impaired insulin sensitivity, affects body weight, adipose tissue (AT) function and circulating metabolic parameters of obesity resistant 129S6 mice in vivo. An obesity-resistant mouse model was chosen to reduce a potential obesity bias of DEHP effects on metabolic parameters and AT function. The metabolic effects of 10-weeks exposure to DEHP were tested by insulin tolerance tests and quantitative assessment of 183 metabolites in mice. Furthermore, 3T3-L1 cells were cultured with DEHP for two days, differentiated into mature adipocytes in which the effects on insulin stimulated glucose and palmitate uptake, lipid content as well as on mRNA/protein expression of key adipocyte genes were investigated. We observed in female mice that DEHP treatment causes enhanced weight gain, fat mass, impaired insulin tolerance, changes in circulating adiponectin and adipose tissue Pparg, adiponectin and estrogen expression. Serum metabolomics indicated a general increase in phospholipid and carnitine concentrations. In vitro, DEHP treatment increases the proliferation rate and alters glucose uptake in adipocytes. Taken together, DEHP has significant effects on adipose tissue (AT) function and alters specific serum metabolites. Although, DEHP treatment led to significantly impaired insulin tolerance, it did not affect glucose tolerance, HOMA-IR, fasting glucose, insulin or triglyceride serum concentrations. This may suggest that DEHP treatment does not cause impaired glucose metabolism at the whole body level.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143190PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4668085PMC
June 2016

Validation of a multi-analyte HPLC-DAD method for determination of uric acid, creatinine, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid and 2-methylhippuric acid in human urine.

J Chromatogr B Analyt Technol Biomed Life Sci 2015 Aug 24;998-999:40-4. Epub 2015 Jun 24.

Department of Metabolomics, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany. Electronic address:

During the last decades exposure sciences and epidemiological studies attracts more attention to unravel the mechanisms for the development of chronic diseases. According to this an existing HPLC-DAD method for determination of creatinine in urine samples was expended for seven analytes and validated. Creatinine, uric acid, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid, and 2-methylhippuric acid were separated by gradient elution (formate buffer/methanol) using an Eclipse Plus C18 Rapid Resolution column (4.6mm×100mm). No interfering signals were detected in mobile phase. After injection of blank urine samples signals for the endogenous compounds but no interferences were detected. All analytes were linear in the selected calibration range and a non weighted calibration model was chosen. Bias, intra-day and inter-day precision for all analytes were below 20% for quality control (QC) low and below 10% for QC medium and high. The limits of quantification in mobile phase were in line with reported reference values but had to be adjusted in urine for homovanillic acid (45mg/L), niacinamide 58.5(mg/L), and indole-3-acetic acid (63mg/L). Comparison of creatinine data obtained by the existing method with those of the developed method showing differences from -120mg/L to +110mg/L with a mean of differences of 29.0mg/L for 50 authentic urine samples. Analyzing 50 authentic urine samples, uric acid, creatinine, hippuric acid, and 2-methylhippuric acid were detected in (nearly) all samples. However, homovanillic acid was detected in 40%, niacinamide in 4% and indole-3-acetic acid was never detected within the selected samples.
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http://dx.doi.org/10.1016/j.jchromb.2015.06.021DOI Listing
August 2015

The lasting effect of limonene-induced particle formation on air quality in a genuine indoor environment.

Environ Sci Pollut Res Int 2015 Sep 14;22(18):14209-19. Epub 2015 May 14.

Department of Urban and Environmental Sociology, Helmholtz Centre for Environmental Research - UFZ, Permoserstrasse 15, 04318, Leipzig, Germany,

Atmospheric ozone-terpene reactions, which form secondary organic aerosol (SOA) particles, can affect indoor air quality when outdoor air mixes with indoor air during ventilation. This study, conducted in Leipzig, Germany, focused on limonene-induced particle formation in a genuine indoor environment (24 m(3)). Particle number, limonene and ozone concentrations were monitored during the whole experimental period. After manual ventilation for 30 min, during which indoor ozone levels reached up to 22.7 ppb, limonene was introduced into the room at concentrations of approximately 180 to 250 μg m(-3). We observed strong particle formation and growth within a diameter range of 9 to 50 nm under real-room conditions. Larger particles with diameters above 100 nm were less affected by limonene introduction. The total particle number concentrations (TPNCs) after limonene introduction clearly exceed outdoor values by a factor of 4.5 to 41 reaching maximum concentrations of up to 267,000 particles cm(-3). The formation strength was influenced by background particles, which attenuated the formation of new SOA with increasing concentration, and by ozone levels, an increase of which by 10 ppb will result in a six times higher TPNC. This study emphasizes indoor environments to be preferred locations for particle formation and growth after ventilation events. As a consequence, SOA formation can produce significantly higher amounts of particles than transported by ventilation into the indoor air.
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http://dx.doi.org/10.1007/s11356-015-4663-8DOI Listing
September 2015

Monitoring of drug intake during pregnancy by questionnaires and LC-MS/MS drug urine screening: evaluation of both monitoring methods.

Drug Test Anal 2015 Aug 28;7(8):695-702. Epub 2014 Dec 28.

Department of Metabolomics, Helmholtz Centre for Environmental Research -UFZ, Leipzig, Germany.

Various studies pointed towards a relationship between chronic diseases such as asthma and allergy and environmental risk factors, which are one aspect of the so-called Exposome. These environmental risk factors include also the intake of drugs. One critical step in human development is the prenatal period, in which exposures might have critical impact on the child's health outcome. Thereby, the health effects of drugs taken during gestation are discussed controversially with regard to newborns' disease risk. Due to this, the drug intake of pregnant women in the third trimester was monitored by questionnaire, in addition to biomonitoring using a local birth cohort study, allowing correlations of drug exposure with disease risk. Therefore, 622 urine samples were analyzed by an untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) urine screening and the results were compared to self-administered questionnaires. In total, 48% (n = 296) reported an intake of pharmaceuticals, with analgesics as the most frequent reported drug class in addition to dietary supplements. 182 times compounds were detected by urine screening, with analgesics (42%; n = 66) as the predominantly drug class. A comparison of reported and detected drug intake was performed for three different time spans between completion of the questionnaires and urine sampling. Even if the level of accordance was low in general, similar percentages (~25%, ~19%, and ~ 20%) were found for all groups. This study illustrates that a comprehensive evaluation of drug intake is neither achieved by questionnaires nor by biomonitoring alone. Instead, a combination of both monitoring methods, providing complementary information, should be considered.
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http://dx.doi.org/10.1002/dta.1767DOI Listing
August 2015

Lefetamine-derived designer drugs N-ethyl-1,2-diphenylethylamine (NEDPA) and N-iso-propyl-1,2-diphenylethylamine (NPDPA): metabolism and detectability in rat urine using GC-MS, LC-MSn and LC-HR-MS/MS.

Drug Test Anal 2014 Oct 3;6(10):1038-48. Epub 2014 Mar 3.

Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, D-66421, Homburg (Saar), Germany.

N-Ethyl-1,2-diphenylethylamine (NEDPA) and N-iso-propyl-1,2-diphenylethylamine (NPDPA) are two designer drugs, which were confiscated in Germany in 2008. Lefetamine (N,N-dimethyl-1,2-diphenylethylamine, also named L-SPA), the pharmaceutical lead of these designer drugs, is a controlled substance in many countries. The aim of the present work was to study the phase I and phase II metabolism of these drugs in rats and to check for their detectability in urine using the authors' standard urine screening approaches (SUSA). For the elucidation of the metabolism, rat urine samples were worked up with and without enzymatic cleavage, separated and analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS). According to the identified metabolites, the following metabolic pathways for NEDPA and NPDPA could be proposed: N-dealkylation, mono- and bis-hydroxylation of the benzyl ring followed by methylation of one of the two hydroxy groups, combinations of these steps, hydroxylation of the phenyl ring after N-dealkylation, glucuronidation and sulfation of all hydroxylated metabolites. Application of a 0.3 mg/kg BW dose of NEDPA or NPDPA, corresponding to a common lefetamine single dose, could be monitored in rat urine using the authors' GC-MS and LC-MS(n) SUSA. However, only the metabolites could be detected, namely N-deethyl-NEDPA, N-deethyl-hydroxy-NEDPA, hydroxy-NEDPA, and hydroxy-methoxy-NEDPA or N-de-iso-propyl-NPDPA, N-de-iso-propyl-hydroxy-NPDPA, and hydroxy-NPDPA. Assuming similar kinetics, an intake of these drugs should also be detectable in human urine.
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http://dx.doi.org/10.1002/dta.1621DOI Listing
October 2014

Subtoxic and toxic concentrations of benzene and toluene induce Nrf2-mediated antioxidative stress response and affect the central carbon metabolism in lung epithelial cells A549.

Proteomics 2013 Nov 16;13(21):3211-21. Epub 2013 Oct 16.

Helmholtz Centre for Environmental Research, Department of Proteomics, Leipzig, Germany.

Since people in industrialized countries spend most of their time indoors, the effects of indoor contaminants such as volatile organic compounds become more and more relevant. Benzene and toluene are among the most abundant compounds in the highly heterogeneous group of indoor volatile organic compounds. In order to understand their effects on lung epithelial cells (A549) representing lung's first line of defense, we chose a global proteome and a targeted metabolome approach in order to detect adverse outcome pathways caused by exposure to benzene and toluene. Using a DIGE approach, 93 of 469 detected protein spots were found to be differentially expressed after exposure to benzene, and 79 of these spots were identified by MS. Pathway analysis revealed an enrichment of proteins involved in Nrf2-mediated and oxidative stress response glycolysis/gluconeogenesis. The occurrence of oxidative stress at nonacute toxic concentrations of benzene and toluene was confirmed by the upregulation of the stress related proteins NQO1 and SOD1. The changes in metabolism were validated by ion chromatography MS/MS analysis revealing significant changes of glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate, and NADPH. The molecular alterations identified as a result of benzene and toluene exposure demonstrate the detrimental effect of nonacute toxic concentrations on lung epithelial cells. The data provided here will allow for a targeted validation in in vivo models.
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http://dx.doi.org/10.1002/pmic.201300126DOI Listing
November 2013

Identification of new protein coding sequences and signal peptidase cleavage sites of Helicobacter pylori strain 26695 by proteogenomics.

J Proteomics 2013 Jun 9;86:27-42. Epub 2013 May 9.

Department of Proteomics, UFZ, Helmholtz-Centre for Environmental Research Leipzig, 04318 Leipzig, Germany.

Unlabelled: Correct annotation of protein coding genes is the basis of conventional data analysis in proteomic studies. Nevertheless, most protein sequence databases almost exclusively rely on gene finding software and inevitably also miss protein annotations or possess errors. Proteogenomics tries to overcome these issues by matching MS data directly against a genome sequence database. Here we report an in-depth proteogenomics study of Helicobacter pylori strain 26695. MS data was searched against a combined database of the NCBI annotations and a six-frame translation of the genome. Database searches with Mascot and X! Tandem revealed 1115 proteins identified by at least two peptides with a peptide false discovery rate below 1%. This represents 71% of the predicted proteome. So far this is the most extensive proteome study of Helicobacter pylori. Our proteogenomic approach unambiguously identified four previously missed annotations and furthermore allowed us to correct sequences of six annotated proteins. Since secreted proteins are often involved in pathogenic processes we further investigated signal peptidase cleavage sites. By applying a database search that accommodates the identification of semi-specific cleaved peptides, 63 previously unknown signal peptides were detected. The motif LXA showed to be the predominant recognition sequence for signal peptidases.

Biological Significance: The results of MS-based proteomic studies highly rely on correct annotation of protein coding genes which is the basis of conventional data analysis. However, the annotation of protein coding sequences in genomic data is usually based on gene finding software. These tools are limited in their prediction accuracy such as the problematic determination of exact gene boundaries. Thus, protein databases own partly erroneous or incomplete sequences. Additionally, some protein sequences might also be missing in the databases. Proteogenomics, a combination of proteomic and genomic data analyses, is well suited to detect previously not annotated proteins and to correct erroneous sequences. For this purpose, the existing database of the investigated species is typically supplemented with a six-frame translation of the genome. Here, we studied the proteome of the major human pathogen Helicobacter pylori that is responsible for many gastric diseases such as duodenal ulcers and gastric cancer. Our in-depth proteomic study highly reliably identified 1115 proteins (FDR<0.01%) by at least two peptides (FDR<1%) which represent 71% of the predicted proteome deposited at NCBI. The proteogenomic data analysis of our data set resulted in the unambiguous identification of four previously missed annotations, the correction of six annotated proteins as well as the detection of 63 previously unknown signal peptides. We have annotated proteins of particular biological interest like the ferrous iron transport protein A, the coiled-coil-rich protein HP0058 and the lipopolysaccharide biosynthesis protein HP0619. For instance, the protein HP0619 could be a drug target for the inhibition of the LPS synthesis pathway. Furthermore it has been proven that the motif "LXA" is the predominant recognition sequence for the signal peptidase I of H. pylori. Signal peptidases are essential enzymes for the viability of bacterial cells and are involved in pathogenesis. Therefore signal peptidases could be novel targets for antibiotics. The inclusion of the corrected and new annotated proteins as well as the information of signal peptide cleavage sites will help in the study of biological pathways involved in pathogenesis or drug response of H. pylori.
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http://dx.doi.org/10.1016/j.jprot.2013.04.036DOI Listing
June 2013

Studies on the metabolism and toxicological detection of glaucine, an isoquinoline alkaloid from Glaucium flavum (Papaveraceae), in rat urine using GC-MS, LC-MS(n) and LC-high-resolution MS(n).

J Mass Spectrom 2013 Jan;48(1):24-41

Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, D-66421, Homburg, Saar, Germany.

Glaucine ((S)-5,6,6a,7-tetrahydro-1,2,9,10-tetramethoxy-6-methyl-4H-dibenzo [de,g]quinoline) is an isoquinoline alkaloid and main component of Glaucium flavum (Papaveraceae). It was described to be consumed as recreational drug alone or in combination with other drugs. Besides this, glaucine is used as therapeutic drug in Bulgaria and other countries as cough suppressant. Currently, there are no data available concerning metabolism and toxicological analysis of glaucine. To study both, glaucine was orally administered to Wistar rats and urine was collected. For metabolism studies, work-up of urine samples consisted of protein precipitation or enzymatic cleavage followed by solid-phase extraction. Samples were afterwards measured by liquid chromatography (LC) coupled to low or high-resolution mass spectrometry (HR-MS). The phase I and II metabolites were identified by detailed interpretation of the corresponding fragmentations, which were further confirmed by determination of their elemental composition using HR-MS. From these data, the following metabolic pathways could be proposed: O-demethylation at position 2, 9 and 10, N-demethylation, hydroxylation, N-oxidation and combinations of them as well as glucuronidation and/or sulfation of the phenolic metabolites. For monitoring a glaucine intake in case of abuse or poisoning, the O- and N-demethylated metabolites were the main targets for the gas chromatography-MS and LC-MS(n) screening approaches described by the authors. Both allowed confirming an intake of glaucine in rat urine after a dose of 2 mg/kg body mass corresponding to a common abuser's dose.
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http://dx.doi.org/10.1002/jms.3112DOI Listing
January 2013

Biofilm inhibition by an experimental dental resin composite containing octenidine dihydrochloride.

Dent Mater 2012 Sep 18;28(9):974-84. Epub 2012 May 18.

Clinic of Operative Dentistry, Periodontology and Preventive Dentistry, Saarland University Hospital, Homburg/Saar, Germany.

Objectives: The aim of the present study was to investigate an antimicrobial additive containing experimental resin composite with regards to its impact on biofilm formation under oral conditions.

Methods: Biofilms were established in situ on composite specimens (n=192) which contained octenidine dihydrochloride (ODH, 3 wt.% or 6 wt.%). Samples without antimicrobial additive served as control (n=96). Composite specimens were fixed on custom made splints and exposed to the oral cavity of six healthy volunteers for three or seven days. Biofilm formation was assessed by scanning electron microscopy (SEM) and fluorescence microscopy (FM).

Results: The biofilm formation was significantly reduced on ODH containing samples compared to controls after three as well as after seven days in situ. FM evaluation additionally showed a lower viability of the reduced biofilms for both ODH concentrations.

Significance: During this short term investigation, incorporation of ODH into resin based composite materials caused biofilm inhibiting effects in situ.
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http://dx.doi.org/10.1016/j.dental.2012.04.034DOI Listing
September 2012