international conference healthcare integrated biobanking ... · 2014 conference program version...

International Conference

“Healthcare integrated biobanking and multiomics biomarker analysis”

July 3–5, 2014

Lecture hall A2 University Hospital Regensburg

Franz-Josef-Strauß-Allee 11 D-93053 Regensburg, Germany

2014 CONFERENCE PROGRAM Version 01.07.2014 13:07

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Day 1: July 3, 2014

(Time schedule includes a discussion of 5 minutes)

13:00 – 13:10 Introduction

Gerd Schmitz (Regensburg, Germany)

Session 1: Health-care integrated biobanking

Chairs: Michael Kiehntopf (Jena, Germany) and Michael Neumaier (Mannheim, Germany)

13:10 – 13:35 Biobanking in multi-omics studies: lessons from cardiovascular diseases"

Thomas Illig (Hannover, Germany)

13:35 – 14:00 Biobanking and metabolomics from newborn screening to health-care

Joachim Thiery (Leipzig, Germany)

14:00 – 14:25 Biobanking and targeted metabolomics for discrimination of systemic inflammatory disorders in critically ill patients

Michael Kiehntopf (Jena, Germany)

14:25 – 14:50 Cryostability of analytes in long term biobanking

Michael Neumaier (Mannheim, Germany)

14:50 – 15:15 Healthcare integrated Biobanking-The Regensburg project

Susanne Heimerl (Regensburg, Germany)

15:15 – 15:45 Coffee break

Session 2: Transcriptomics and epigenetics

Chairs: Assam El-Osta (Melbourne, Australia) & Antonio Moschetta (Bari, Italy)

15:45 – 16:10 The opportunities and caveats of multigenic nuclear receptor regulation as therapeutic targets

Antonio Moschetta (Bari, Italy)

16:10 – 16:35 Epigenetic control by histone methylation

Andrew J. Pospisilik (Freiburg, Germany)

16:35 – 17:00 Epigenetic implications for the diabesity syndrome

Assam El-Osta (Melbourne, Australia)

17:00 – 17:25 The role of telomeres in cellular aging: effect of vitamins supplementation on telomere length and LINE-methylation in elderly

Irene Pusceddu (Homburg, Germany)

17:25 – 17:45 Shotgun lipidomics and histology of the human lung: a perfect couple to gain insight into metabolic perturbation caused by pathological processes

Dominik Schwudke (Borstel, Germany)

17:45 – open end: Poster session with wine & cheese

Healthcare integrated biobanking and multiomics biomarker analysis


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Day 2: July 4, 2014

Session 3: Proteomics in civilization disorders

Chairs: Helmut Meyer (Dortmund, Germany) & Jens Wiltfang (Göttingen, Germany)

08:30 – 08:55 FIBRO-4 as a novel Biomarker for Liver Fibrosis and Cirrhosis

Helmut Meyer (Dortmund, Germany)

08:55 – 09:20 Protein biomarkers in neurodegenerative diseases

Jens Wiltfang (Göttingen, Germany)

09:20 – 09:45 Alpha-synuclein and related proteins in the pathogenesis of Parkinson’s disease

Jochen Klucken (Erlangen, Germany)

09:45 – 10:10 MRM-based plasma proteomics in microbial infection and inflammation

Christoph H. Borchers (Victoria, Canada)

10:10 – 10:30 Afamin – a novel marker for the prevalence and incidence of metabolic syndrome and related diseases?

Hans Dieplinger (Innsbruck, Austria)


Session 4: Membrane microdomains and extracellular vesicles

Chairs: Elina Ikonen (Helsinki, Finland) & Anette Draeger (Bern, Switzerland)

11:00 – 11:25 Live imaging of cholesterol-rich domains in normal and cholesterol storage disease cells

Elina Ikonen (Helsinki, Finland)

11:25 – 11:50 Extracellular vesicles in inflammation and autoimmunity

Edit Buzás (Budapest, Hungary)

11:50 – 12:15 The “annexin-survival package”: plasma membrane repair and cellular damage control

Anette Draeger (Bern, Switzerland)

12:15 – 12:40 Platelet-derived extracellular vesicles: implications to platelet transfusion and diseases

Anne Black (Regensburg, Germany)

12:40 – 12:55 Anti-atherogenic effects of sphingosine 1-phosphate (S1P) mediated by modulation of macrophage, T-cell and endothelial functions

Jerzy-Roch Nofer (Muenster, Germany)

13:00 – 14:00 Lunch break


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Day 2: July 4, 2014, (continued)

Session 5: Organelle lipidomics

Chairs: Rudolf Zechner (Graz, Austria) & Michael Schlame (New York, USA)

14:00 – 14:25 Lipid droplets and diabesity

Rudolf Zechner (Graz, Austria)

14:25 – 14:50 Endolysosomal lipids and phospholipidosis

Evelyn Orsó (Regensburg, Germany)

14:50 – 15:15 Antiphospholipid antibodies – can cellular phospholipids be pathogenetically relevant targets?

Karl Lackner (Mainz, Germany)

15:15 – 15.40 Turnover measurements of mitochondrial lipids in Barth syndrome

Michael Schlame (New York, USA)

15:40 – 15:55 Peroxisomal function in health and disease

Ronald J. A. Wanders (Amsterdam, The Netherlands)

15:55 – 16:15 Refreshments

Keynote lecture

Chair: Wolfgang Stremmel (Heidelberg, Germany)

16:15 – 16:45 High-density lipoproteins: from reverse cholesterol transport to extracellular

vesicle release and remodelling in vascular disease

Gerd Schmitz (Regensburg, Germany)

19:00 – open end: Gala dinner


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Day 3: July 5, 2014

Session 6: Lipidomics in vascular and metabolic diseases

Chairs: Christian Wolfrum (Zürich, Switzerland) & Ira J. Goldberg (New York, USA)

08:30 – 08:55 Marker of vascular and metabolic diseases: lessons from population genetics

Heribert Schunkert (Munich, Germany)

08:55 – 09:20 Major players of cellular lipid traffic in obesity: from mouse to man

Christian Wolfrum (Zürich, Switzerland)

09:20 – 09:45 Oxysterols and non-alcoholic fatty liver disease

Luigi Iuliano (Latina, Italy)

09:45 – 10:10 Lipidomic “cascade” screening of plasma sterols: diagnostic stratification and therapeutic monitoring

Silke Matysik (Regensburg, Germany)

10:10 – 10:30 The effect of n-3 PUFA on markers of insulin resistance in prediabesity

Aldona Dembinska-Kiec (Krakow, Poland)


Session 7: Cellular stress management & lipotoxicity

Chairs: Laszlo Vigh (Szeged, Hungary) & Anna Nicolaou (Manchester, UK)

11:00 – 11:25 Short-lived signalling lipids in inflammation

Anna Nicolaou (Manchester, UK)

11:25 – 11:50 Many pathways to LIPO-tox the heart

Ira J. Goldberg (New York, USA)

11:50 – 12:15 Lipid microdomains, ER stress and autophagy in cancer cell therapy

Pablo V. Escribá (Palma de Mallorca, Spain)

12:15 – 12:35 Lipids, as key players in cellular heat stress management

Gábor Balogh (Szeged, Hungary)

12:35 – 12:55 The role of milk signalling in mTORC1-driven diseases of civilization: from artificial infant nutrition to persistent milk consumption

Bodo Melnik (Gütersloh, Germany)

13:00 Closing remarks


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Abstracts (oral presentations)

The role of telomeres in cellular aging: effect of vitamins supplementation on telomere length and LINE-methylation in elderly

Irene Pusceddu, Wolfgang Herrmann, Susanne Kirsch, Rima Obeid, Ulrich Hübner, Jürgen Geisel and Markus Herrmann1

Department of Clinical Chemistry, Saarland University Hospital, Germany; 1Department of Clinical Pathology, District Hospital Bolzano, Italy

Background: Telomeres are essential for the maintenance of genomic integrity. Telomere length

declines with age and telomere shortening/dysfunction has been proposed as biomarker for age-

related diseases. B and D vitamins are essential cofactors for numerous cellular processes

including the synthesis of purines and nucleotides, DNA methylation, cell differentiation,

proliferation and apoptosis. B and D vitamin deficiencies are risk factors for the development of

age-related diseases. The aim of this study was to evaluate the effects of B and D vitamin

supplementation on telomere biology in healthy elderly people.

Methods: In a double-blind study 60 subjects (>54 years) were randomly assigned to receive a

daily combination of vitamin D3 (1200 IU), folic acid (0.5 mg), vitamin B12 (0.5 mg), vitamin B6 (50

mg) and calcium carbonate (456 mg) (Group A) or vitamin D3 and calcium carbonate alone (Group

B). Blood concentrations of 25-hydroxy-vitamin D, vitamin B12, vitamin B6, folate and several

metabolites were measured. Furthermore, LINE-methylation and telomere length in peripheral

blood leucocytes were analyzed at baseline and after 1 year of supplementation.

Results: Baseline gender- and age-adjusted telomere length correlated with methyl-

tetrahydrofolate (THF; r=0,35), 5,10-methenyl-tetrahydrofolate (r=0,36) and total folate (r=0,33). At

the end of the study gender- and age-adjusted telomere length showed the following correlations:

Group A: metylmalonic acid (r=-0,46) and choline (r=0,39); Group B: 5,10-methenyl-

tetrahydrofolate (r=-0,57), dimethyl-glycine (r=-0,39), and LINE-1 methylation (r=-0,43).

Conclusions: The present results suggest a functional relationship between vitamin B status and

telomere length in elderly subjects. Moreover, a sub-optimal vitamin B status may result in

telomere dysfunction through altered DNA methylation.

Shotgun lipidomics and histology of the human lung: a perfect couple to gain insight into metabolic perturbation caused by pathological processes.

Julia. Müller2, Verena Scholz1, T. Goldmann2,3 and D. Schwudke1,3

1 Division of Bioanalytical Chemistry - Research Center Borstel, Parkallee 1-40, 23845 Borstel, Germany; 2 Division of Clinical and Experimental Pathology - Research Center Borstel, Parkallee 1-40, 23845 Borstel, Germany; 3 Airway Research Center North – German Center for Lung Research, Wöhrendamm 80, 22927 Grosshansdorf, Germany

Relatively little is known about the lung lipidome and how it influences the maintenance of the

barrier functions and gas exchange. With this study we lay the basis for studying the lipid metabolic

perturbations, which can be associated with chronic obstructive pulmonary disease (COPD).

We have established a shotgun lipidomics workflow for the analysis of different tissue and cell

types of the human lung. Alveolar, bronchial and cancer tissue biopsies were divided for

histological characterization using the HOPE method and sample preparation for lipidomics. Lipids

were extracted with a customized methyl tert-butyl ether / methanol based extraction method.

Lipidomics screens were performed using an Apex Qe FTICR-MS mass spectrometer (Bruker

Daltonics, Bremen, Germany) equipped with a TriVersa NanoMate robot (Advion, Ithaca, USA).


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Lipid identification was performed with the LipidXplorer software accessing quantitative information

of approximately 250 membrane and neutral lipids.

Between the different tissues, complex profile changes were observed which we start to associate

with age, gender and pathology. First lipidomics results indicate that the quantity of typical

surfactant lipids in alveolar tissues can directly be used for functional associations. We could show

using multivariate data analysis of lipidomics data and a histological scoring system that main

phosphatidylcholine (PC) and phosphaditylglycerol (PG) and sphingomyelin (SM) lipids are

potential marker for the health of alveolar tissues while increasing amounts of triacylglycerols

(TAG) indicate pathological processes.

This pilot study is the starting point to learn more about the influence of the lipid metabolism in the

progression of lung diseases like COPD.

Afamin – a novel marker for the prevalence and incidence of metabolic syndrome and related diseases?

Hans Dieplinger

Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Austria

Background. Afamin is a human plasma glycoprotein with vitamin E binding properties primarily

expressed in the liver and secreted into the bloodstream. Since very little is known about

physiological or pathophysiological functions of afamin, we investigated transgenic mice

overexpressing the human afamin gene and performed large-scale human epidemiological studies

to identify phenotypes and possible functions associated with afamin.

Methods and Results. Transgenic mice revealed increased body weight and serum

concentrations of lipids and glucose. The investigated human cohorts were the population-based

Bruneck Study (n=826), the SAPHIR Study (n=1499), and the KORA F4 Study (n=3060). We

applied a fixed-effects meta-analysis using baseline (available in all three cohorts) and 5-year

follow-up investigations (available in Bruneck and SAPHIR). Mean afamin concentrations were

62.5±15.3, 66.2±14.3, and 70.6±17.2 mg/L in Bruneck, SAPHIR and KORA F4, respectively. We

observed a 18% higher probability for an increase of one metabolic syndrome component per 10

mg/L increase of afamin at baseline (p=2.17*10-230) and an 8% higher probability for a gain in one

further component between baseline and follow-up (p=8.87*10-16). Afamin concentration at

baseline was highly significantly related to all single metabolic syndrome components at baseline

and follow-up. This observation was most pronounced for an elevated waist circumference

(OR=1.72, p=5.79*10-123 at baseline and OR=1.46, p=2.84*10-11 for a change during follow-up)

and elevated fasting glucose concentrations (OR=1.46, p=1.87*10-69, and OR=1.47, p=1.01*10-

18, respectively).

Conclusions. This study in more than 5,000 participants shows that afamin is strongly associated

with the prevalence and development of metabolic syndrome and all its components. Supported by

results from transgenic mice and incidence data in human populations, a high potential for afamin

as predictive marker for the metabolic syndrome and related diseases such as obesity, diabetes

and steatosis can be assumed. Our results from transgenic animals overexpressing human afamin

suggest causality of these findings with possible therapeutic implications.


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Anti-atherogenic effects of sphingosine 1-phosphate (S1P) mediated by modulation of macrophage, T-cell and endothelial functions

Jerzy-Roch Nofer

Center for Laboratory Medicine, University Hospital Münster, Münster, Germany

Sphingosine 1-phosphate (S1P) is a bioactive lysosphingolipid and a constituent of high density

lipoprotein (HDL) responsible for several atheroprotective effects of HDL exerted in vitro. However,

studies on anti-atherogenic effects of S1P in vivo are scarce and led to disparate results. We

previously demonstrated that synthetic S1P analogues such as FTY720 - a pan-specific S1P

receptor agonist and KRP203 – a specific S1P receptor type 1 (S1PR1) agonist reduced

atherosclerosis in cholesterol-rich diet-fed LDL-R-deficient (LDL-R-/-) mice. Anti-atherogenic

effects exerted by both S1P analogues were accompanied by skewing T-cell and macrophage

polarization towards less inflammatory Th2 and M2 phenotypes, respectively, as evidenced by

-like receptor 3 and 4 agonists as well as increased production of

anti-inflammatory mediators such as IL-1RA and prostaglandin E2. In the ensuing studies we

focused on anti-atherogenic effects exerted by endogenous S1P and mediated via modulation of

endothelial function. To this purpose, sublethaly irradiated LDL-R-/- mice were transplanted with

bone marrow (BM) obtained from sphingosine kinase 2 (SphK2)-deficient mice, which led to

elevation of plasma S1P concentration by approximately 1.6 – 2.0 folds. Afterwards animals were

fed cholesterol-rich diet for 14 weeks. In parallel experiments, KRP203 was administered

intraperitoneally to LDL-R-/- mice on cholesterol-rich diet. Both, elevation of endogenous S1P and

treatment with exogenous S1P analogue decreased cholesterol accumulation in arterial wall.

Furthermore, monocyte recruitment to atherosclerotic lesions and lipopolysaccharide (LPS)-

induced recruitment to peritoneal cavity were both reduced in SphK2-/--BM-transplanted and in

KRP203-treated LDL-R-/- mice. Studies using intravital microscopy revealed that both endogenous

S1P and exogenous S1P analogue lowered leukocyte adhesion to capillary wall and decreased

endothelial permeability to fluorescently labeled LDL. Moreover, decreased plasma levels of

soluble adhesion molecules (sVCAM-1, sICAM-1) were observed in animals after SphK2-/-

transplantation or KRP203 treatment. Studies in vitro showed that both S1P and KRP203 reduced

monocyte adhesion to and transport across endothelial layer. In addition, decreased permeability

to fluorescence-lab Session eled dextran beads or LDL was observed in S1P- or- KRP203-treated

endothelial cells. Similar effects on endothelial function were observed in cells exposed to murine

plasma enriched in S1P or plasma obtained from SphK2-deficient animals. It is concluded that both

endogenous S1P and exogenous synthetic S1P analogues exert anti-atherogenic effects that are

mediated by favorable modulation of macrophage, T-cell and endothelial function.

The effect of n-3 PUFA on markers of insulin resistance in prediabesity 1Polus A., 2Kieć-Wilk B., 1Malczewska-Malec M., 1Fedak D., 3 Sanak M., 4Calder P., 1Dembinska-Kiec A

1Chair and Department of Clinical Biochemist, 2Chair and Department of Metabolic Disease, 3Division of Molecular Biology and Clinical Genetics,II Chair of Internal Medicine; The Jagiellonian

University, Krakow, Poland, and 4Human Development & Health Academic Unit Faculty of

Medicine University of Southampton; Southampton General Hospital,UK,

Background: Inflammation is now recognized as a causative event for atherosclerosis, diabetes,

cancer, autoimmune diseases etc. The return from inflammation to the “non-inflamed status”

(catabasis) is an actively regulated program of resolution. Introduction of LC-MS/MS has provided

information of the new class PUFA derived lipid autacoids (i.e. lipoxins generated from ω-6 AA and

resolvins, protectins, maresins generated from ω-3 PUFA), with the specific anti-inflammatory and


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proresolving activity. Recent studies pointed out that γ-carboxylation of osteocalcin (Gla-OC) are

connected with reduced insulin resistance associating low grade inflammation in prediabesity.

Aim: The aim of the study was to investigate the effect of ω-3 PUFA on biosynthesis of

osteocalcin; resolvins as well as the low grade inflammatory markers and insulin sensitivity

Materials and methods: Patients aged 25-65 years with BMI>25-40 kg/m2 assigned to two groups

with: n-3 PUFA supplementation (EPAX1050TG -1, 8 g DHA/EPA (5:1)) per day for 3 months or

with placebo supplementation (corn oil) in frame of randomized study. All patients before and after

3 months of PUFA / placebo supplementation undergone the 2h oral glucose(OGTT) and 8h oral

lipid (OLTT) tolerance test. Glucose, insulin, incretin GIP, carboxylated (Gla-OC),

undercarboxylated (Glu-OC) osteocalcin and resolvins/lipoxins (LC-MS/MS) were measured and

correlated with the inflammatory markers (hsCRP, MPC-1, E-selectin)..Effect of exogenous PUFAs

on resolving biosynthesis by differentiating human preadipocytes was measured in in vitro model.

Results: The level of Gla-OC was increased in patients under ω-3 PUFA. Supplementation

decreased serum glucose and insulin in 60 min during OGTT, which resulted also in decreased

insulin AUC. Negative associations between Gla-OC and some markers of insulin sensitivity:

glucose and GIP output during OGTT as well as glucose levels during OLTT (r = 0,2 <0,05) was

found. Analysis of subgroups revealed that obesity counteracts shift towards carboxylated (Gla-

OC) form of osteocalcin (OC). Negative association of Gla-OC/Glu-OC ratio with sE-selectin level

as well as weak positive correlation of undercarboxylated osteocalcin (Glu-OC) plasma level with

leptin concentration (r = 0,2 p<0,05) was observed. Incubation of differentiating human

preadipocytes with PUFAs, as well as the EPAX supplementation of patients resulted in

incorporation of ω-PUFA into cellular phospholipids and increase of anti-inflammatory rersolvins

extracellular concentration.

Conclusions: Three months of ω-3 PUFA supplementation (DHA/EPA 5:1) increased the

carboxylated osteocalcin level (Gla-OC), increased the resolvin biosynthesis when decreased the

markers of inflammation It may add to the understanding of the mechanism of the tissue insulin

sensitivity protection in prediabetes by marine oils, serving as the new biomarkers of this process..

Supported by: EU FP7 BIOCLAMS, Grant agreement no. 244995 grant K/ZDS/002442 and NCN

MAESTRO grant no. K/PBN/000001.

Lipids, as key players in cellular heat stress management

Gábor Balogh, Mária Péter, Attila Glatz, Péter Gudmann, Bálint Csoboz, Imre Gombos, Zsolt Török, Ibolya Horváth and László Vígh

Institute of Biochemistry, Biological Research Centre, Hung. Acad. Sci. H-6726 Szeged, Hungary

Earlier we reported how membrane composition could modulate the stress response. The

numerous sensors at the top of the pathways are interconnected by the parameters of the

chemical and physical state of membrane. Membrane alterations, signal pathways from

membranes to heat shock protein (Hsp) genes and Hsps themselves play fundamental roles in the

aetiology of several human diseases, such as type 2 diabetes and cancer. Improving the cellular

stress response via Hsp induction provided protection against insulin resistance after consuming a

high fat diet, and was coupled with reduced fat stores in mice. Interestingly, acute heat stress is

able to activate lipid droplet (LD) formation in different cell lines. Moreover, in the HSF1 -/- MEF

cells, where the heat-inducibility of Hsps is almost completely abrogated, the LD formation was

largely enhanced upon thermal stress.


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To gain insight into interconnection between stress response, membrane-lipid signalling and LD

biogenesis, the yeast model Schizosaccharomyces pombe was studied. High resolution shotgun

mass spectrometric analysis revealed that S. pombe responded to heat stress with profound

reorganisation of its whole lipidome. In addition to the bilayer stabilizing compositional shift of

membrane lipids, enhanced production of signal lipids (e.g. certain ceramides, phosphatidic acid

species and diglyceride), and the storage lipid triglyceride (TG) were observed. In the trehalose

synthase yeast mutant, where trehalose – that has been shown to stabilize proteins and

membranes during heat shock – cannot be formed, the stress-induced TG synthesis and the signal

lipids generation were more pronounced than in wild-type cells. Furthermore, the almost totally TG-

synthesis deficient (dga1/plh1) mutant S. pombe cells displayed cell cycle arrest and striking

elevation of diglyceride level upon heat stress.

Consequently, LDs may have a key role in the stress-regulation of lipid mediator and membrane

lipid metabolism. Moreover, we suggest that LDs may have some unexpected “holdase”-like

function as protective reservoirs for unfolded proteins formed due to stress.


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Abstracts (poster presentations)

DNA methylation in smokers and never smokers with different FTO genotypes.

Jaroslav A. Hubáček1, Dana Dlouhá1

1 Center for Experimental Medicine, Institute for Experimental Medicine, Vídeňská 1958/9, 14021, Prague, Czech Republic;

Background: Cigarette smoking is the most common form of tobacco use and is a major

preventable cause of cancer and cardiovascular disease. Level of DNA methylation is an important

predictor of many biochemical procedures within the human body and is a result of the both

environmental and genetic factors. Recent genome wide association studies have identified

variants within the FTO gene (possible DNA demethylase activity) to influence risk of the wide

range of the noncommunicable diseases.

Aim: To evaluate the total DNA methylation in individuals with different smoking status and different FTO first intron tagging variants.

Methods: Forty two current smokers and 46 never smokers (all Caucasians, non-diabetics) with

equally distributed FTO genotypes (in each group 50% of GG and TT homozygotes for the 1st

intron tagging variant rs17817449) with similar age and BMI were included in the study.

MethylFlashTM Methylated DNA Quantification Kit Colorimetric (Epigentek Group Inc., Farmingdale,

NY, USA) was used to analyse the total DNA methylation status.

Results: Total DNA methylation was slightly higher in the entire group of smokers if compared with

never smokers (6.39±4.62 5mC% vs. 4.56±2.14 5mC%, P = 0.049). However, FTO genotypes

were not associated with total DNA methylation neither within the entire group (P = 0.78), nor

within the smokers (P = 0.87) or never smokers (P = 0.94).

Conclusion: Our pilot study suggests, that the total DNA methylation is dependent on the smoking

status, but not on the 1st intron FTO genotypes. Mechanism, how FTO influences the risk of

obesity, diabetes and cardiovascular diseases needs to be further studied.

Acknowledgements: This study was supported by the project No. NT/12170-5 (IGA, MH CR).

Lipidomic Analysis of Serum from High Fat Diet Induced Obese Mice

Sabrina Krautbauer1, Kristina Eisinger1, Gerhard Liebisch2, Gerd Schmitz2, Charalampos Aslanidis2 and Christa Buechler1

1 Department of Internal Medicine I, Regensburg University Hospital, 93042 Regensburg, Germany; 2 Institute for Clinical Chemistry and Laboratory Medicine, Regensburg University Hospital, 93042 Regensburg, Germany

Background: Obesity is a primary risk factor for metabolic diseases. Lipid metabolites regulate fatty

acid and glucose homeostasis. Lipidomics techniques demonstrate a high complexity of the

plasma lipidome. These methods are used to identify new lipid biomarkers associated with obesity

and type 2 diabetes which may be relevant in pathophysiology, diagnosis and therapy.

Aim: The intention of the current study is to identify circulating lipid species, which are altered in

rodent obesity and strongly correlate with the metabolites glucose, triglycerides and cholesterol.

Methods: Male mice were fed a high fat diet for 14 weeks. Lipids in the serum were quantified by

direct flow injection electrospray ionization tandem mass spectrometry (ESI-MS/MS) in positive ion

mode. were measured in serum. Proinsulin and insulin levels were determined by ELISA.

Triglycerides, cholesterol and glucose concentrations were analyzed with commercial kits.


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Results: Mice fed a high fat diet have increased body weight and fasting glucose. Serum

triglycerides are not altered, while cholesterol tends to be increased. Accordingly, major cholesteryl

ester (CE) species and free cholesterol are not significantly raised in obesity while minor

metabolites are increased or reduced, respectively. Distinct sphingomyelin species are elevated

while ceramides are not raised. Some phosphatidylinositol (PI) species are raised while others are

decreased. PI 34:1 strongly correlates with fasting glucose and proinsulin levels.

Phosphatidylcholine (PC) 26:0, 40:2, and 40:5, which are induced in obesity, correlate with

cholesterol. PC 38:4 and PC 40:6 are also raised in obese mice and positively correlate with

fasting glucose. Lysophosphatidylcholine (LPC) species are also changed in obesity and the

already shown reduction of LPC 16:1 has been confirmed. Increased LPC 22:4 correlates with

serum cholesterol.

Conclusion: The data indicate that circulating levels of various lipid species are changed in the

obesity model studied and some of them are strongly associated with classically measured

metabolites.

Lipidomic analysis of the liver from high-fat diet induced obese mice identifies changes in multiple lipid classes

Kristina Eisinger1, Sabrina Krautbauer1, Tobias Hebel1, Gerd Schmitz2, Charalampos Aslanidis2, Gerhard Liebisch2 and Christa Buechler1

1 Department of Internal Medicine I, Regensburg University Hospital, 93042 Regensburg, Germany; 2 Institute for Clinical Chemistry and Laboratory Medicine, 93042 Regensburg University Hospital, Regensburg, Germany

Background: The prevalence of overweight and obesity has dramatically increased and is closely

associated with the rising prevalence of non-alcoholic fatty liver disease (NAFLD). More than 50%

of obese people have liver steatosis which may progress to non-alcoholic steatohepatitis (NASH)

and liver cirrhosis. Recent studies have applied lipidomic techniques for in-depth analysis of the

hepatic lipids in NAFLD. Numerous changes in the hepatic lipid composition have been identified.

Aim: Intention of the current study is 1) to identify lipid species changed in rodent fatty liver, 2) to

analyze for possible associations of these lipids with triglycerides, cholesterol or CXCL8 which is

elevated in the steatotic liver and 3) to find out whether systemic levels of these lipids are

concordantly altered.

Methods: Male mice were fed a high fat diet or a standard diet for 14 weeks. Lipids in the liver were

quantified by direct flow injection electrospray ionization tandem mass spectrometry (ESI-MS/MS)

in positive ion mode. Gene expression was analyzed by real-time RT-PCR and protein expression

by SDS-PAGE and immunoblotting.

Results: Lipidomic analysis has confirmed already reported reduction of phosphatidylcholine in the

steatotic liver. Phosphatidylserine is lower and phosphatidylethanolamine tends to be diminished.

Sphingomyelin levels are normal while monounsaturated ceramides and hexosylceramides are

reduced. Sixteen of the 20 fatty acid species measured in the total lipid fraction are elevated while

-linolenic acid is diminished. Medium chain saturated fatty acids are markedly decreased.

Plasmalogens 18:0 and 18:1 species are strongly increased in the steatotic liver. None of the

markedly changed individual lipid species strongly correlates with hepatic CXCL8 mRNA,

triglycerides or cholesterol. About 60% of the lipids altered in fatty liver are congruently altered in

serum.

Conclusion: These data show that there are multiple changes in hepatic lipid composition and part

of the lipids may be monitored by serum analysis.


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Adiponectin isoforms differentially affect gene expression and lipidome of primary human hepatocytes

Lisa Voggenreiter1, Josef Wanninger1, Gerhard Liebisch2, Kristina Eisinger1, Markus Neumeier1, Rebekka Pohl1, Charalampos Aslanidis 2, Thomas S. Weiss 3

, Sabrina Krautbauer1 and Christa Buechler1

1 Department of Internal Medicine I, Regensburg University Hospital, 93042 Regensburg, Germany; 2 Institute for Clinical Chemistry and Laboratory Medicine, Regensburg University Hospital, 93042 Regensburg, Germany; 3 University Children Hospital (KUNO), Regensburg University Hospital, 93042 Regensburg, Germany

Background: Obesity is a major risk factor for metabolic diseases concerning the liver like non-

alcoholic fatty liver disease (NAFLD). Adiponectin (APN) is an adipokine which is secreted by

adipose tissue and functions as an anti-inflammatory, anti-steatotic and anti-fibrotic agent which

protects from liver injury. APN also counteracts adverse metabolic effects in obesity but APN levels

in the serum are found to be reduced in obese individuals. There are different APN isoforms

circulating in the serum including low molecular weight (LMW-APN) and high molecular weight

(HMW-APN), which show different biological effects.

Aim: Aim of the study was to analyze the effect of low molecular weight (LMW) and higher

molecular weight (HMW) APN on primary human hepatocytes (PHH).

Methods: PHH were incubated with LMW and HMW APN for 24 h. Gene expression was analyzed

by real-time RT-PCR. Activin A, follistatin and apolipoprotein B-100 were measured by ELISA.

Lipids were quantified by direct flow injection electrospray ionization tandem mass spectrometry

(ESI-MS/MS) in positive ion mode.

Results: APN is not detected in hepatocyte lysates. Levels are, however, strongly increased by

HMW-APN but not by LMW-APN, suggesting distinct uptake / degradation of APN isoforms by

PHH. Several genes with a role in fibrosis, glucose and lipid metabolism known to be regulated by

HMW-APN are not affected by the LMW isoform. Follistatin is reduced by HMW-APN and induced

by LMW-APN in supernatants of PHH. Fibroblast growth factor 21 mRNA is, however, repressed

by both isoforms. Cellular triglycerides and cholesterol levels are not reduced by APN. Total

phospholipids including plasmalogens and sphingomyelins are not changed upon APN incubation

while distinct species are either induced or repressed. Unexpectedly, total ceramide is increased

by LMW-APN.

Conclusion: Current data show that APN isoforms differentially affect hepatocyte function but do

not grossly alter hepatocyte lipidome.

Chiral separation of aliphatic hydroxycarboxylic acids using liquid and supercritical fluid chromatography

Zoltán Pataj1, Federica Ianni2,3, Harald Gross3, Wolfgang Lindner4, Michael Lämmerhofer3

1 Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary; 2 Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy; 3 Institute of Pharmaceutical Sciences, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany; 4 Department of Analytical Chemistry, University of Vienna, Währinger Strasse 38, 1090 Vienna, Austria

While aliphatic 2-hydroxyalkanoic acids have been more or less successfully enantioseparated

with various chiral stationary phases by HPLC and GC, analogous applications on underivatized

aliphatic 3-hydroxyalkanoic acids are completely absent in the scientific literature. With the aim of

closing this gap, the enantioseparation of 3-hydroxybutyric acid, 3-hydroxydecanoic acid and 3-


[15]

hydroxymyristic acid has been performed by HPLC with two ion-exchange type chiral stationary

phases (CSPs): one containing the anion-exchange type tert-butyl carbamoyl quinine chiral

selector motif (Chiralpak QN-AX), and the other carrying the new zwitterionic variant based on

trans-(S,S)-2-aminocyclohexanesulfonic acid-derivatized quinine carbamate (Chiralpak ZWIX(+))

as the chiral selector and enantiodiscriminating element, respectively. Moreover the effects of

organic modifier and temperature on the enantioseparation of 6 hydroxycarboxylic acids (2- and 3-

hydroxybutyric acid, 3-hydroxydecanoic, 2-hydroxyglutaric acid, tartaric acid and malic acid) using

supercritical fluid chromatography (SFC) have been investigated. Chiralpak ZWIX(+) and Chiralpak

ZWIX(-) based on trans-(R,R)-2-aminocyclohexanesulfonic acid derivatized quinine carbamate,

were explored for the chiral separation of these compounds.

During the HPLC measurements, the zwitterionic enantiorecognition material provided better

results in terms of enantioselectivity and resolution compared to the anion-exchanger CSP at

reduced retention times due to the intramolecular counterion effect imposed by the sulfonic acid

moiety and its competition with the 3-hydroxyalkanoic acid analyte for ionic interaction at the

quininium-anion exchanger site. It is thus recommended as the CSP of first choice for

enantioseparations of the class of aliphatic 3-hydroxyalkanoic acids. Hence chiral zwitterionic-

exchangers were exclusively used to achieve the enantioseparation of various hydroxycarboxylic

acids by SFC. The major experimental variables have been investigated for optimization of the

resolution and allowed to derive information on the enantiorecognition mechanism in both cases.

The elution order of the enantiomers was found to be reversal on the pseudo-enantiomeric

Chiralpak ZWIX(+) and Chiralpak ZWIX(-) columns. The recommended methods are MS

compatible.

Acknowledgement: The research by Z. P. was carried out in the frame of TAMOP 4.2.4. A/2–11–1–

2012–0001 “National Excellence Program—Elaborating and operating an inland student and

researcher personal support system convergence program. The project was subsidized by the

European Union and cofinanced by the European Social Fund.

Chemerin induces cell death of hepatocyte cell lines in-vitro and in an in-vivo xenograft model

Rebekka Pohl1, Sabrina Krautbauer1, Kristina Eisinger1, Michael Beck1, Laura Eichelberger1, Thomas S. Weiss2 and Christa Buechler1

1 Department of Internal Medicine I, Regensburg University Hospital, 93042 Regensburg, Germany; 2 University Children Hospital (KUNO), Regensburg University Hospital, 93042 Regensburg, Germany

Background: Non-alcoholic fat liver disease (NAFLD) is the most common liver disease in western

population and is often found in obese individuals where NAFLD represents the hepatic

manifestation of the metabolic syndrome. Chemerin is highly expressed in the liver and its

concentration in the serum shows a moderate positive correlation with the body mass index.

Accordingly, the levels of chemerin in the serum of morbidly obese patients with non-alcoholic

steatohepatitis (NASH) is increased.

Aim: Aim of the current study was to find out whether active chemerin (Chem-157) affects

hepatocytes.

Methods: Primary human hepatocytes and stellate cells, HepG2 and Huh7 cells were incubated

with increasing concentrations of Chem-157 for 24 h. Affymetrix whole-transcript Human Gene 2.1

ST Array was hybridized with RNA isolated from primary liver cells. Nude mice were


[16]

subcutaneously inoculated with HepG2 cells. Chem-157 was injected in the tumour from day 7 to

day 21 three times a week.

Results: Chemerin and its receptor CMKLR1 were found to be expressed by primary human

hepatocytes, HepG2 and Huh7 cells. Incubation of primary human hepatocytes with Chem-157 did

not significantly affect gene expression. Furthermore, viability of the primary cells was not reduced

by Chem-157. Unexpectedly, Chem-157 induced death of the hepatocyte cell lines HepG2 and

Huh7. Injection of Chem-157 in the tumour did not affect viability of the mice nor serum chemerin

levels. Importantly, tumour weight of the HepG2 xenograft was significantly reduced.

Conclusion: Chem-157 does not affect gene expression and viability of primary human

hepatocytes but induces cell death of hepatocyte cell lines in-vitro and HepG2 cells in-vivo.

Differentiation of mycobacterium species by shotgun lipidomics

Nicole Zehethofer1, Ulrich E. Schaible1, Dominik Schwudke2

1 Research Center Borstel, Cellular Microbiology, Parkallee 26, Borstel, Germany; 2 Research Center Borstel, Bioanalytical Chemistry, Parkallee 10, Borstel, Germany

Mycobacterium tuberculosis (M. tb), the causative agent of Tuberculosis, was responsible for 1.4

Million deaths in 2011. Mycobacterial cell wall lipids are unique among prokaryotes and comprise

crucial factors of mycobacterial virulence and pathogenicity. Current protocols for global

characterization of the M. tb lipidome rely upon time-consuming lipid class separation prior to mass

spectrometric analysis using thin layer chromatography or liquid chromatography. Here we present

a rapid and reproducible protocol for profiling mycobacterial lipids by stepwise fractionation of lipids

according to their hydrophobicity during extraction. The described method was successfully applied

for mycobacterium species differentiation (M. tb and M. marinum).

Bacterial cells (M. tb H37Rv and M. marinum, 10 ml cultures) were extracted using a petroleum

ether/methanol mixture. Analyses of the lipid extracts were performed in the positive and negative

ion mode using an Apex Qe FTICR-MS equipped with a Triversa Nanomate nano-ESI source.

Mass spectra were subsequently smoothed, baseline subtracted and centroidized using a

customized VBA script (Data Analysis 4.0). Lipids were identified by their monoisotopic masses

using LipidXplorer applying a mass accuracy better than 5ppm. Molecular fragmentation query

language files for mycobacterial lipid identification were compiled according to the entries of the

MtbLipidDB database.

The established method permitted identification of more than 600 complex lipid species in

mycobacterial lipid extracts such as phosphatidylinositol mannosides, sulfolipids, and TDM in a

rapid (5 min/sample) and reproducible (SD<10 %) manner. Distinct differences were identified in

the lipid signatures of M. tb and M. marinum showing that stepwise extraction of lipids is a valuable

tool to distinguish mycobacterial species based on their lipid signatures.

Future work will focus on the correlation of mycobacterial lipid signatures with virulence of clinically

relevant strains.

Acknowledgements: We would like to thank Kristine Hagens, Jacqueline Eich, Michael Weinkauf,

and Steffen Pichlo for excellent technical assistance as well as Monika Hagedorn and Lilli

Gerstenmaier for providing the M. marinum strain.


[17]

MALDI imaging MS reveals candidate lipid markers of polycystic kidney disease

Hermelindis Ruh1,2,3,4, Theresia Salonikios2,5, Jens Fuchser6, Matthias Schwartz2,5, Bernhard Wirnitzer2,5, Norbert Gretz2,3,4 and Carsten Hopf1,2,3*

1 Instrumental Analysis and Bioanalysis, Department of Biotechnology; 2 Center for Applied

Research in Biomedical Mass Spectrometry (ABIMAS), Mannheim University of Applied Sciences,

Paul-Wittsack-Str. 10, 68163 Mannheim, Germany; 3 Institute of Medical Technology, University of

Heidelberg and Mannheim University of Applied Sciences; 4 Medical Research Center, Theodor-

Kutzer-Ufer 1-3, 68167 Mannheim, Germany; 5 Institute of Digital Signal Processing, Mannheim

University of Applied Sciences, Paul-Wittsack-Str. 10, 68163 Mannheim, Germany; 6 Bruker

Daltonik GmbH, Fahrenheitstr. 4, D-28359 Bremen, Germany

Autosomal recessive polycystic kidney disease (ARPKD) is a severe childhood-onset, hepatorenal

fibrocystic disease (HRFCD)1. It is characterized by the progressive formation of massive renal

cysts and by biliary dysgenesis resulting in congenital hepatic fibrosis. Up to now, no simple

diagnostic test for ARPKD is available. As clinical manifestations in ARPKD vary in renal and

hepatic involvement and as morphological changes might not be indicative for a single type of

HRFCD, the identification of body fluid-derived markers that could distinguish ARPKD from other

HRFCDs is urgently needed.

Alterations in lipid profiles in several kidney diseases suggest that defined subsets of lipids may be

useful as molecular disease markers2-3. We therefore set out to establish a workflow for

identification of candidate lipid makers by matrix-assisted laser desorption/ionization (MALDI)

imaging mass spectrometry (IMS) of relevant tissues. However, applicable workflows that link

MALDI lipid IMS and identification as well as structural characterization of candidate disease

classifying marker lipids are lacking.

Here, we combine selective MALDI IMS of sulfated kidney lipids employing a method that our

laboratory has published recently4, and Fisher discriminant analysis of imaging data sets for

identification of candidate markers of progressive disease in PCK rats.

Our study highlights strong increases in distinct lipids as main classifiers of cystic disease.

Structure determination by high resolution Fourier Transform (FT)-MS identifies these altered lipids

as taurine-conjugated bile acids. These sulfated lipids are selectively elevated in the PCK rat

model but not in models of related HRFCDs suggesting that they be molecular markers of the

disease.

A combination of MALDI IMS with high resolution FT-MS methods and Fisher discriminant data

analysis may be widely applicable for lipid marker discovery.

Acknowledgements:

This work was supported by the Baden-Württemberg Ministry of Science and Culture (INST 874/2-

1 LAGG to C.H.) and by a joined grant (“ZAFH ABIMAS”) from ZO IV by the Landesstiftung Baden-

Württemberg and the Europäischer Fonds für regionale Entwicklung (EFRE; to N.G., B.W., and

C.H.).

(1) Gunay-Aygun, M., Liver and kidney disease in ciliopathies. Am J Med Genet C Semin Med Genet 2009, 151C (4), 296-306.

(2) Natoli, T. A.; Husson, H.; Rogers, K. A.; Smith, L. A.; Wang, B.; Budman, Y., . . . Ibraghimov-Beskrovnaya, O., Loss of GM3 synthase gene, but not sphingosine kinase 1, is protective against murine nephronophthisis-related polycystic kidney disease. Hum Mol Genet 2012, 21 (15), 3397-407.


[18]

(3) Mather, A. R.; Siskind, L. J., Glycosphingolipids and kidney disease. Adv Exp Med Biol 2011, 721, 121-38.

(4) Marsching, C.; Eckhardt, M.; Grone, H. J.; Sandhoff, R.; Hopf, C., Imaging of complex sulfatides SM3 and SB1a in mouse kidney using MALDI-TOF/TOF mass spectrometry. Anal Bioanal Chem 2011, 401 (1), 53-64.

Two-dimensional ultra high-performance liquid chromatography – mass spectrometry characterization of comprehensive samples of human plasma

Magdaléna Ovčačíková1, Eva Cífková1, Miroslav Lísa1, Michal Holčapek1

1University of Pardubice, Faculty of Chemical Technology, Department of Analytical Chemistry, Studentská 573, 53210 Pardubice, Czech Republic

Lipids belong among one of the most important constituents forming all biological tissues and body

fluids. Their interaction with surrounding cells has a crucial role in living organisms. The changes of

lipid metabolism play a significant role in many serious human diseases, such as cancer,

atherosclerosis, obesity, diabetes mellitus, cardiovascular disease and Alzheimer's disease.

Lipidomics is a subset of metabolomics, which contributes to the understanding how lipids work in

a biological system (cellular physiology and pathology) [1-3]. One-dimensional chromatographic

separation may be not sufficient for the detailed lipidomic characterization due to various types of

isomerism. Therefore two-dimensional ultra high-performance liquid chromatography - mass

spectrometry (2D-UHPLC/MS) can be used to increase the number of compounds separated and

quantitated in a single run [3, 4]. Our work is focused on the development of a new comprehensive

2D-UHPLC method with electrospray ionization mass spectrometry (ESI-MS) for the lipidomic

characterization of complex biological samples. As a first dimension, reversed-phase liquid

chromatography (RP-LC) with a C18 column is used, where lipids are separated according to the

acyl chain length and the number of double bonds. Hydrophilic interaction liquid chromatography

(HILIC) mode using a silica column is used as the second dimension. In HILIC separation mode,

lipids are separated into individual lipid classes according to their polarity and electrostatic

interactions. Fractions from the first dimension column are transferred to the second dimension

column using a two-position eight-port switching valve, operated under on-line conditions in

combination with MS detection.

Acknowledgement

This work was supported by ERC CZ grant project LL1302 (Ministry of Education, Youth and

Sports of the Czech Republic).

References

[1] Fahy, E., Subramaniam, S., Brown, H. A., Glass, C. K., et al., Eur. J. Lipid Sci. Technol. 2005, 107, 337-364.

[2] van Meer, G., Voelker, D. R., Feigenson, G. W., Nat. Rev. Mol. Cell Biol. 2008, 9, 112-124.

[3] Lísa, M., Cífková, E., Holčapek, M., J. Chromatogr. A 2011, 1218, 5146-5156.

[4] Cífková, E., Holčapek, M., Lísa, M., Ovčačíková, M., et al., Anal. Chem. 2012, 84, 10064-10070.


[19]

Determination of Prostacyclin and Thromboxane A2 urine metabolites by LC-MS/MS method in hypertensive, NO-deficient mice

Agnieszka Kij1, Maria Walczak1,2, Łukasz Mateuszuk1, Barbara Sitek1, Krystyna Wandzel1, Stefan Chłopicki1,3

1Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University,

Bobrzyńskiego 14, 30-348 Kraków, Poland; 2Department of Pharmacokinetics and Physical Pharmacy, Jagiellonian University, Medical College, Medyczna 9, 30-688 Kraków, Poland; 3Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University, Medical College, Grzegórzecka 16, Kraków, Poland

Background: Eicosanoids are implicated in many physiological and pathological processes.

In particular COX dependent AA–derived prostanoids including prostaglandins (PGs), prostacyclin

(PGI2) and thromboxane A2 (TXA2) are involved in vascular homeostasis.

Aim: The goal of this work was to develop a bioanalytical methods for quantification of TXA2

metabolites (TXB2, 2,3-dinor-TXB2 and 11-dehydro-TXB2), PGI2 metabolites (2,3-dinor-6-keto-

PGF1α, 6-keto-PGF1α) and other prostanoids (tetranor-PGEM, tetranor-PGDM, 11β-PGF2α,

PGE2, PGD2, PGF2α) as well as isoprostanes (8-isoPGF2α) in mice urine and/or plasma using

LC-MS/MS technique in order to characterize changes in their generation in C57Bl/6J mice with

NO-deficiency and hypertension induced by L-NAME. Furthermore, the blood pressure was

measured using cuff-tail method. Platelet activation was evaluated by ex vivo dynamic TXB2

generation assay.

Methods: Quantification of selected eicosanoids was accomplished employing ultrafast liquid

chromatograph UFLC Nexera (Shimadzu) coupled to mass spectrometer QTRAP 5500 (ABSciex)

equipped with TurboV ion source. The limit of quantification for TXA2, PGI2 and PGs metabolites

in urine was approximately 250 pg/mL, 500 pg/mL and 500 pg/mL, respectively. In case of PGs,

the quantification limit in plasma was approximately 10ng/mL. Urine and plasma samples were

prepared by employing liquid-liquid extraction with acidified ethyl acetate, which provided the best

purification efficiency between tested techniques (e.g. protein precipitation with and without

evaporation step, solid-phase extraction). TXB2 concentration after platelet activation was

determined using an enzyme-linked immunosorbent assay (ELISA) kit.

Results and conclusions: Development of L-NAME induced hypertension was associated with

transient overactivation of platelets as evidenced by higher ex vivo TXB2 generation in L-NAME

treated mice as compared to control mice in 2nd week but not 4th weeks of L-NAME treatment. On

the other hand, the excretion of urinary 2,3-dinor-TXB2 as well as 2,3-dinor-6-keto-PGF1α was

increased along L-NAME treatment. Presented results suggest the prolonged but not short-term

NO-deficiency induced a compensatory PGI2 overproduction that resulted in blunting of platelet

overactivation induced by impaired NO function. The model of L-NAME induced hypertension

seems well suited for COX-2/PGI2 pathway pharmacology in vivo.

Acknowledgements: This work was supported by European Union from the resources of the

European Regional Development Fund under the Innovative Economy Programme (grant

coordinated by JCET-UJ, No POIG.01.01.02-00-069/09.


[20]

Comprehensive lipidomic profiles of CVD patients studied by UHPLC/MS and MALDI-MS

Blanka Červená1, Eva Cífková1, Miroslav Lísa1, Vitaliy Chagovets1, Michal Holčapek1, Jitka Vostálová2, Jan Galuszka3, Martin Hill4

1 University of Pardubice, Studentská 573, 53210 Pardubice, Czech Republic; 2 Palacký University Olomouc, Tř. Svobody 8, 77126 Olomouc, Czech Republic, 3 Faculty Hospital Olomouc, I.P. Pavlova 185/6, 77900 Olomouc, Czech Republic, 4 Institute of Endocrinology, Národní 8, 11694 Prague 1, Czech Republic

Lipids play a key role in the metabolism of human organism and their dysregulation could lead to

onset of cardiovascular diseases (CVD), which are one of the most common causes of mortality in

the world. Main risk factors are age, gender, hypertension, smoking, physical inactivity, diabetes,

hyperhomocysteinemia, obesity, atherosclerosis and high level of circulating lipids. Due to lipid

roles in many biological functions, reliable analytical methods for their determination are essential.

Polar lipids (phosphatidylcholines, phosphatidylethanolamines, sphingomyelines and

lysophosphatidylcholines) were measured using hydrophilic interaction liquid chromatography in

ultrahigh-performance liquid chromatography (UHPLC) setup coupled with electrospray ionization

mass spectrometry. Normal-phase UHPLC coupled with atmospheric pressure chemical ionization

mass spectrometry was used for the identification of nonpolar lipids (cholesteryl esters, cholesterol,

triacylglycerols, diacylglycerols and monoacylglycerols). Lipid classes were quantified using

methods with a single internal standard and response factors (sphingosyl phosphatidylethanolmine

d17:1/12:0 for polar lipids and 1,2-dioleoyl ethylene glycol for nonpolar lipids) [1]. Matrix-assisted

laser desorption/ionization mass spectrometry was used for the fast analysis of total lipid extracts

of plasma and erythrocytes. No statistically significant differences were observed in the quantity of

polar and nonpolar lipid classes probably due to their large biological variability. The orthogonal 2

projection to latent structure (O2PLS) method was used for the determination of 5 groups (healthy,

obese and 3 types of CVD). O2PLS was used for the separation of the healthy volunteers group

vs. CVD patients group and statistically important compounds were identified. The main aim of our

study is to highlight dynamic changes of lipids in CVD and differences between patients and

healthy volunteers. Our findings could be useful in future search of lipid biomarkers of CVD and

contribute to earlier diagnostic or treatment of CVD.

Acknowledgement:

This work was supported by the grant project No. 206/11/0022 (Czech Science Foundation).

Reference:

1. Cífková E, Holčapek M, Lísa M, Ovčačíková M, Lyčka A, Lynen F, Sandra P, Anal. Chem 84 (2012) 10064-10070.

Comparison of LC/ESI-MS and ESI-MS analyses of oxidized 1-palmitoyl-2-arachidonoyl-sn-

glycero-3-phosphocholine products on human lipoproteins

Mine-Yine Liu*, You-Sian Lin

Department of Chemistry, National Changhua University of Education, Changhua 50058, Taiwan

Background: 1-palmitoyl-2- arachidonoyl-sn-glycero-3-phosphocholine (PAPC) is a very important

phospholipid on human lipoproteins. Oxidized PAPC products are pro-inflammatory and bioactive.

The amounts of oxidized PAPC (ox-PAPC) products are huge, and many of them have not been

studied yet.


[21]

Aim: LC/ESI-MS and ESI-MS analyses have been compared for the analysis of oxidized PAPC

products on human lipoproteins.

Methods: Fenton reaction has been used to oxidize human high-density lipoprotein (HDL), low-

density lipoprotein (LDL) and very low-density lipoprotein (VLDL). Liquid-liquid and solid-phase

extractions have been used to isolate total lipids and phospholipids from the oxidized human

lipoproteins. The isolated phospholipids were then studied by LC/ESI-MS and ESI-MS analyses.

Results: A total of 9 long-chain and 10 short-chain ox-PAPC products have been observed. The

ion intensities of ox-PAPC products were the highest for Fenton reaction with Fe2+: H2O2 = 2:1.

Conclusion: The direct analysis of ox-PAPC products by ESI-MS method showed better sensitivity

and reproducibility than the LC/ESI-MS method.

Acknowledgements: The authors would like to acknowledge the financial support of the Ministry

of Science and Technology of Taiwan (Grant number: NSC 100-2113-M-018-001-MY3).

Monitoring of characteristic biomarkers in Smith-Lemli-Opitz syndrome (SLOS)

Anna V Oláh1, Gabriella P Szabó2, József Varga3, János Harangi4, István Balogh1

1Departments of Laboratory Medicine, 2Pediatrics, 3 Nuclear Medicine, Medical Center University of Debrecen, 4Research Laboratory for Chromatography, Nagyerdei krt. 98, Debrecen, Hungary H-4032

Background: Fifteen Hungarian children were diagnosed with SLOS, an inherited disorder of

cholesterol biosynthesis. Deficiency of 7-dehydrocholesterol-reductase (DHCR7) decreases

cholesterol and increases dehydrocholesterol (DHC), causing severe somatomental disorders.

Aims: Since genotypes and phenotypes only slightly correlate, relation between lipid markers and

clinical score was tested. As the liver function is affected by dehydrocholesterols, we monitored the

liver function during cholesterol supplementation and Statin therapy.

Patients and results: Their age at the time of diagnosis was 0.5-18 years in mild type SLOS (n=4,

clinical score <20), cholesterol was 2.37±0.8 mmol/L, 7DHC 147±55 mg/L. In typical SLOS

cholesterol was 1.47±0.7 mmol/L, 7DHC=202±77 mg/L (n=7, score:20-50, age: 0.1-7 years).

Patients with severe SLOS had the lowest cholesterol (0.66±0.27 mmol/L, 7DHC=181±52 mg/L),

all died as newborn (clinical score>50, n=4). In severe SLOS percentage of α-lipoprotein was lower

than in typical (p=0.003) and mild SLOS (p=0.004). Decreased ratio of α-lipoproteins detected in

severe SLOS compared to the control group may be the consequence of cholesterol biosynthesis

disorder. Increase of transaminases (AST, ALT) in 50% of patients during cholesterol

supplementation (n=10) combined with statin therapy (n=9), refers to a reversible damage of liver

in typical and severe SLOS.

Conclusion: Our data show the prognostic value of initial cholesterol, 7DHC, HDL-C. They

determine severity of SLOS and life expectancy. Simvastatin treatment in SLOS can’t be

considered as safe therapy, therefore monitoring of lipid parameters and liver function is

recommended. Dehydrocholesterols and their metabolites are toxic to the liver, accumulation of

7DHC inhibits formation of lipoproteins. Lack of HDL-C will slow down the reverse cholesterol

transport, which also reduces cholesterol level. 7DHC has high reactivity with oxygen radicals, it

probably accelerates lipid peroxidation, which is harmful to HDL-C and HDL-associated

antioxidants (e.g. paraoxonase). Although the mechanism needs further research, antioxidant

therapy seems to be promising in SLOS.


[22]

Simultaneous mass spectrometric analysis of cholesterol precursors, phytosterols and

oxysterols in the diagnosis of dyslipidemia

S. Matysik, G. Schmitz

Universitätsklinikum Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany

To assess an exact evaluation of the balance between cholesterol biosynthesis and intestinal

absorption the analysis of cholesterol precursors, i.e. intermediates of endogenous cholesterol

biosynthesis such as lanosterol and lathosterol and phytosterols, such as sitosterol, which serve as

absorption markers is recommended. Ratios between sitosterol/lathosterol can be used to evaluate

the major features of cholesterol metabolism and to differentiate between “oversynthesizers” and

“hyperabsorbers”. By means of this individual sterol profile recommendations can be generated for

hypercholesterolemia patient stratification for therapy. The analysis of sterols also includes the

quantification of oxidative products of cholesterol, the oxysterols. Oxysterols play essential roles in

the regulation of various biological processes and functions exerting a multitude of biological

effects of potential pathophysiological relevance and can therefore be regarded as severity and

progression markers of atherogenesis. Oxysterols like 24(S)-hydroxycholesterol are associated

with Alzheimer’s disease and 7-ketocholesterol and cholestane- -triol are sensitive

biomarkers for Niemann-Pick-Type C. Thus, robust analytical procedures for clinical routine are

required. Gas chromatography coupled to triple quad MS offers new opportunities in the combined

analysis of cholesterol precursors, phytosterols and oxysterols. Main results of recent studies are

(i) statin treatment reduces serum cholesterol precursors but increases serum phytosterols, (ii)

cholesterol precursors are associated with Apo E genotypes, (iii) oral probiotic supplementation

reduces total cholesterol and phytosterols in hypercholesterolemic adults.

Profiling fatty acid synthesis and processing to track cellular fatty acid species

Josef Ecker, Gerhard Liebisch, Max Scherer, Gerd Schmitz


Fatty acids are major components of membrane phospholipids, autacoid precursors and a basic

source of energy for many organisms. Therefore, proper regulation of fatty acid synthesis and

processing is crucial and dysregulation leads to several metabolic diseases. In mammalians, one

key player of fatty synthesis is the fatty acid synthase complex catalyzing the generation of fatty

acids from acetyl-Coenzyme A (CoA). Activation of fatty acids with CoA is key step for metabolism

of these compounds.

To profile total fatty acid composition, we developed a quantitative gas chromatography – mass

spectrometry (GC-MS) method for fatty acid methyl esters (FAMEs). In one run, FAMEs are

separated and analyzed within 15 minutes. The fatty acids species, their positional and cis/trans

isomers, are characterized with a SCAN mode and quantified by a simultaneous single ion

monitoring (SIM) mode detecting the specific fragments of saturated and unsaturated fatty acids.

To study in vivo fatty acid synthesis, we used stable isotope labeled acetate (2-13C-acetate) and

calculated isotope enrichment in fatty acids by mass isotopomer distribution analysis using SIM of

molecule ions. For investigation of fatty acid processing, we used stable isotope labeled palmitate

(D3-palmitate). Elongation to stearate or desaturation to palmitoleate was determined by SIM of

specific fragment ions of the metabolites. To analyze metabolism of activated fatty acids we are

currently establishing a method using LC-MS/MS to quantify acyl-CoAs.

Our developed methods provides a powerful tool to understand regulatory mechanisms involved in

cellular lipid homeostasis.


[23]

Analysis of non-esterified fatty acids in human samples by solid-phase-extraction and gas chromatography/mass spectrometry

Thomas Kopf & Gerd Schmitz


The determination of the fatty acid (FA) profile of lipid classes is essential for lipidomic analysis.

We recently developed a GC/MS-method for the analysis of the FA profile of total FAs, i.e. the

totality of bound and unbound FAs, in any given biological sample (TOFAs). Here we present a

method for the analysis of non-esterified fatty acids (NEFAs) in biological samples, i.e. the fraction

that is present as extractable free fatty acids. Lipid extraction is performed according to Dole using

80/20 2-propanol/n-hexane (v/v), with 0.1% H2SO4. The fatty acid-species composition of this

NEFA-fraction is determined as FAME after derivatization with our GC/MS-method on a BPX

column (Shimadzu). Validation of the NEFA-method presented was performed in human plasma

samples. The validated method has been used with human plasma, cells and tissues, as well as

mammalian body fluids and tissue samples. The newly developed solid-phase-extraction (SPE)-

GC-MS method allows the rapid separation of the NEFA-fraction from a neutral lipid extract of

plasma samples. As a major advantage compared to GC-FID-methods, GC-MS allows the use of

stable isotope labeled fatty acid precursors to monitor fatty acid metabolism.

Fenofibrate treatment of fructose-fed rats increases choline phospholipid synthesis and homocysteine – indications for a “choline-stealing”-mechanism at the expense of homocysteine recycling

Thomas Kopf1, Hans-Ludwig Schaefer2, Mark Broenstrup3, Tatiana Konovalova1, Gerd Schmitz1

1Universitätsklinikum Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany

2 Infraserv GmbH & Co. Höchst KG, Frankfurt,

3Helmholtz Centre for Infection Research,

Braunschweig

Fenofibrate lowers plasma triglycerides via peroxisome proliferator activated receptor α-activation.

As side effects increased levels of creatinine, homocysteine and cystathionine in human

plasma/serum have been reported. Here, we analyzed lipidomic changes upon fenofibrate

treatment of fructose fed rats. Three groups with 6 animals each were defined (control, fructose-fed

and fructose-fed/fenofibrate treated). Male Wistar Unilever Rats were subjected to 10% fructose-

feeding for 20 days. On day 14, fenofibrate treatment (100 mg/kg p.o.) was initiated and

maintained for 7 days. Lipid species were analyzed using mass spectrometry (LC-MS/MS; GC-MS)

in serum at three time points (days 0, 14 and 20) in all three groups. In addition, lipid levels in liver

and intestine were determined after sacrificing the animals.

In the liver increases of phosphatidylcholine species, phosphatidylethanolamines,

phosphatidylinositols, phosphatidylglycerols, lysophosphatidylglycerols and bismono-

acylglycerophosphates were observed upon fenofibrate treatment. In contrast, plasmalogen,

phosphatidylserine, ceramide and phosphatidic acid levels were reduced in rat liver under

fenofibrate treatment. The jejunum exhibited no significant changes in lipid classes upon

fenofibrate treatment. In serum, decreased levels of phosphatidylcholines,

phosphatidylethanolamines, phosphatidylinositols, free cholesterol. The increased methylmalonic

acid, homocysteine and reduced choline levels in serum after fenofibrate treatment, combined with

the increase of glycerophospholipids in liver, suggest a “choline-stealing mechanism” in which

fenofibrate induces cellular choline incorporation into phosphatidylcholine-precursors that

diminishes the methyl group donor pool, thereby hampering homocysteine conversion to

methionine. This is supported by RNA expression data indicating a decrease of choline kinase and

of methylenetetrahydrofolate reductase.


[24]

Influence of Fenofibrate treatment on triacylglycerides, diacylglycerides and fatty acids in fructose fed rats

Thomas Kopf1, Hans-Ludwig Schaefer2, Martin Troetzmueller3, Harald Koefeler3, Mark Broenstrup4, Tatiana Konovalova1, Gerd Schmitz1

1Universitätsklinikum Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany;

2 Infraserv GmbH & Co. Höchst KG, Frankfurt,

3Core Facility Mass Spectrometry, ZMF, Medical

University Graz, Helmholtz Centre for Infection Research, Braunschweig

Fenofibrate (FF) lowers plasma triglycerides via PPARa-activation. Here, we analyzed lipidomic

changes upon FF treatment of fructose fed rats. Three groups with 6 animals each were defined as

control, fructose-fed and fructose-fed/FF treated. Male Wistar Unilever Rats were subjected to 10%

fructose-feeding for 20 days. On day 14, fenofibrate treatment (100 mg/kg p.o.) was initiated and

maintained for 7 days. Lipid species in serum were analyzed using mass spectrometry (ESI-

MS/MS; LC-FT-MS, GC-MS) on days 0, 14 and 20 in all three groups. In addition, lipid levels in

liver and intestine were determined.

Short-chain TAGs increased in serum and liver upon fructose-feeding, while almost all TAG-

species decreased under FF treatment. Long-chain unsaturated DAG-levels (36:1, 36:2, 36:4,

38:3, 38:4, 38:5) increased upon FF treatment in rat liver and decreased in rat serum. FAs,

especially short-chain FAs (12:0, 14:0, 16:0) increased during fructose-challenge. VLDL secretion

increased upon fructose-feeding and together with FA-levels decreased to control levels during FF

treatment. Fructose challenge of de novo fatty acid synthesis through fatty acid synthase (FAS)

may enhance the release of FAs ≤16:0 chain length, a process reversed by FF-mediated PPARa-

activation.

High throughput lipid profiling by tandem mass spectrometry

Gerhard Liebisch, Max Scherer, Alfred Böttcher, Gerd Schmitz


It is known that the molecular composition of lipids has great influence on biological functions by

modulation of cell membrane fluidity and curvature affecting signalling processes as well as activity

of membrane bound enzymes. The analysis of lipid species by conventional methodologies is time

consuming, insensitive and unselective. Therefore electrospray tandem mass spectrometry (ESI-

MS/MS) was applied as a platform for high throughput quantification of lipid species.

So far as possible we quantify lipids by direct flow injection of crude lipid extracts. This way

protocols were established for the quantification of the major glycerophospholipid and sphingolipid

classes as well as cholesterol and cholesteryl ester. If necessary for instance due to low

concentration, isobaric species lipid extracts were analyzed by liquid chromatography coupled to

tandem mass spectrometry (LC-MS/MS). In contrast to most existing methods based on reversed

phase chromatography, we used hydrophilic interaction liquid chromatography (HILIC) allowing co-

elution of lipid species with various chain lengths and their internal standard. This approach was

applied for the quantitation of lysophosphatidic acid and low concentrated sphingolipid metabolites

including sphingoid bases, sphingosine-1-phosphate as well as hexosylceramide and

lactosylceramide. Quantification was achieved by standard addition and data processing was

automated by self-programmed Excel macros.

We applied lipid species quantitation in large clinical studies as well as basic research including

plasma samples, cell culture and tissue homogenates. To get more understanding of plasma lipid

species, lipoproteins were separated by fast performance liquid chromatography (FPLC) and


[25]

subsequently analyzed. Additionally, the use of stable isotope labelled lipid precursor allows a

metabolic profiling of the lipid species.

Taken together, lipid profiling by mass spectrometry provides a powerful tool to discover novel lipid

biomarker in the blood compartment and to unravel mechanisms underlying the regulation of

cellular lipid metabolism.

High throughput quantification and metabolic profiling of lipid species

Gerhard Liebisch, Max Scherer, Josef Ecker, Gerd Schmitz


It is known that the molecular composition of lipids has great influence on biological functions by

modulation of cell membrane fluidity and curvature affecting signalling processes as well as activity

of membrane bound enzymes. Therefore electrospray tandem mass spectrometry (ESI-MS/MS)

was applied as a platform for high throughput quantification of lipid species.

Major glycerophospholipid and sphingolipid classes as well as cholesterol and cholesteryl ester

were quantified by direct flow injection of crude lipid extracts. If necessary e.g. due to low

concentration or isobaric species lipid extracts were analyzed by liquid chromatography coupled to

tandem mass spectrometry (LC-MS/MS). Quantification was achieved by standard addition and

data processing was automated by self-programmed Excel macros. To study major pathways of

glycerophospholipid metabolism, we introduced stable isotope labelled precursor like D9-choline,

D4-ethanolamine and 13C3-serine. ESI-MS/MS analysis allows the quantitative analysis of de-

novo synthesized glycerophospholipid species by specific head group scans. A parallel

measurement of unlabeled species facilitates the analysis of fatty acid remodelling. Additionally,

GC-MS is applied to monitor synthesis and metabolism of fatty acids using stable isotope labelled

acetate (2-13C-acetate) or fatty acids. In summary, the application of stable isotope labels enables

to study the dynamics of the lipid species metabolism.

We applied lipid species quantitation in large clinical studies as well as basic research including

plasma samples, cell culture and tissue homogenates. Taken together, lipid profiling by mass

spectrometry provides a powerful tool to discover novel lipid biomarker in the blood compartment

and to unravel mechanisms underlying the regulation of cellular lipid metabolism.

Quantitative analysis of sphingolipids in cells and plasma by LC-MS/MS

Max Scherer, Kerstin Leuthäuser-Jaschinski, Josef Ecker, Gerd Schmitz, Gerhard Liebisch


Sphingolipids comprises a highly diverse and complex class of molecules that serve not only as

structural components of membranes but also as signalling molecules. These metabolites are

known to act as intracellular and extracellular messengers eliciting important cell functions

including apoptosis, differentiation and chemotaxis. To understand the differential role of

sphingolipids in a regulatory network it is important to use structure-specific and quantitative

methods.

We developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method for

the rapid, simultaneous quantification of sphingolipids and metabolites such as sphingosine,

sphinganine, di- and trimethyl-sphingosine, phyto-sphingosine, sphingosylphosphorylcholine,

sphingosine-1-phosphate, hexosylceramide, lactosyl-ceramide and ceramide-1-phosphate in

different cell types and plasma. Appropriate internal standards were added prior to lipid extraction

and the mass spectrometer was operated in the multiple reaction monitoring. In contrast to most

methods in the literature based on reversed phase chromatography, we used hydrophilic


[26]

interaction liquid chromatography (HILIC) and achieved good peak shape, short analysis times and

most important coelution of the analytes and their respective internal standards. In order to avoid

an overestimation of species concentrations, peak areas were corrected regarding isotope overlap.

Quantification was achieved by standard addition. Data processing was highly automated by the

use of Analyst® and self programmed Excel macros.

Validation was performed according to FDA guidelines. The developed method showed excellent

precision, accuracy, detection limits and robustness. Together with analysis times of 2.2 min and

4.5 min these methods are valuable tools to evaluate the sphingolipid rheostat in the regulation of

cell functions as well as to search for novel diagnostic biomarker in large clinical studies.

Effect of high-density lipoprotein 3 (HDL3) on megakaryopoiesis, platelet senescence and platelet extracellular vesicle (PL-EV) release

Annika Pienimäki-Römer, Katharina Rübsaamen, Alfred Böttcher, Astrid Fischer, Maria Tafelmeier, Max Scherer, Gerhard Liebisch, Evelyn Orsó, Gerd Schmitz

Institute for Laboratory and Transfusion Medicine, University of Regensburg, Germany

Beyond its role in reverse cholesterol transport and cell membrane homeostasis, the HDL-pathway

targets protective effects as antioxidant function, endothelial cell protection, anti-inflammatory and -

thrombotic effects on megakaryopoiesis and platelet senescence, the basis of platelet storage

lesion=PSL in platelet substitution therapy. HDL particles mature by sequential, specific uptake of

cholesterol from peripheral tissues through ABCA1 (in megakaryocytes), ABCG1 and SR-B1

(scavenger receptor B1), mediating bidirectional cholesterol flux in platelets.

The ATP-cassette transporter ABCA1 controls platelet shedding from megakaryocytes and packing

of platelet granules, with abnormalities in genetic ABCA1 deficiency. HDL3/apo A-I induce

megakaryocyte podia and platelet release. The anucleate platelets circulate 8 days in the blood

and among blood cells have a unique lipid composition with 4-fold increase of plasma cholesteryl

ester 18:2 (Leidl et al, 2008; Rübsaamen et al, 2010), taken up as plasma lipoprotein lipid species

into the open canalicular system, comprising 30% of the platelet volume. In contrast, released PL-

EVs enrich signaling lipids and their precursors, like lysophosphatidic acid and plasmalogens

(Pienimäki-Römer et al. submitted).

Activation/senescence induce PL-EV release, a concern in stored platelet concentrates for

transfusion. 5 days HDL3 treatment in novel quadruple mini-platelet bag systems for standard PSL

analysis improve platelet microviscosity, reduce EV release and PSL-induced lipidomic, surface

marker and miRNA expression changes. Summed, plasma HDL3/apo A-I control platelet life cycle:

shedding, viability/function and senescence/EV-release.

High-density lipoproteins (HDL) and the plasticizer DINCH suppress formation of platelet-derived extracellular vesicles

Annika Pienimäki-Römer, Astrid Fischer, Maria Tafelmeier, Evelyn Orsó, Gerd Schmitz

Institute for Clinical Chemistry and Transfusion Medicine, University of Regensburg, Germany

Human platelets are anucleated cells with a short lifespan of 8-10 days in the circulation.

Approximately 70–90% of extracellular vesicles in circulating blood are derived from activated or

senescent platelets, developing a platelet storage lesion (PSL). Excessive formation and release of

platelet-derived EVs (PL-EVs) is critical for vascular repair and a major problem in stored platelet

apheresis products for transfusion. We introduced a novel quadruple mini-platelet bag system with

low shear-stress (PVC-DINCH foil) to improve the current in vitro platelet storage procedures. In

comparison with other bag materials, platelets stored in PVC-DINCH bag show impaired PSL and


[27]

reduced formation of PL-EVs. Beyond its role in reverse cholesterol transport and cell membrane

homeostasis, HDL exert protective effects towards anti-oxidant, anti-inflammatory and anti-

thrombotic effects, beneficial for megakaryopoiesis, platelet function and platelet senescence. In

vitro, HDL3/apo A-I induce extensive pro-platelet podia formation and ABCA1-dependent platelet

shedding from megakaryocytes. In stored platelets HDL3 improve platelet membrane

microviscosity and antagonize platelet activation, PL-EV release, and modulate senescence-

dependent changes in platelet lipidomics, expression of surface markers and micro RNAs

(miRNA). Taken together, the low shear-stress PVC-DINCH bag combined with HDL3 treatment

significantly suppress release of PL-EVs, correlating negatively with the degree of HDL-particle

oxidation, and improve platelet viability.

Platelet extracellular vesicles (PL-EVs) are carriers of proteins involved in vascular- and neurodegenerative disease

Gerd Schmitz Annika Pienimäki-Römer, Katja Kuhlmann, Tatiana Konovalova, Alfred Böttcher, Evelyn Orsó, Gerhard Liebisch

Institute for Laboratory Medicine and Transfusion Medicine, University of Regensburg, Germany

Introduction: During activation and senescence, platelets release increased amounts of PL-EVs.

We established an in vitro model for proteomic and lipidomic characterization of platelets, PL-EVs

and surrounding plasma over 5 days in platelet concentrates.

Methods: After 5 days standard blood banking, PL-EVs were isolated by filtration and differential

gradient ultracentrifugation into 5 subfractions (F1-F5), and subjected to proteomic and lipidomic

mass spectrometry. In addition small RNA´s were determined.

Results: F1-F2 express 50% of endolysosomal transport from which 25% are ESCRT-proteins

(ALIX, CD36, CD9), 45% of RNA-binding proteins (including Argonaute), platelet activation

markers CD62P, Annexin V, and the Parkinson-related protein alpha-synuclein. F2-F4 express

CD63 and LAMP2. The Alzheimer protein amyloid beta precursor protein (APP) resides in F3-F4.

F3-5 enrich vascular remodeling proteins caveolin-1, apolipoproteins Apo AI ,-J and -E. F5

contains 42% mitochondrial proteins.

Conclusions: PL-EV subfractions show differential expression of the neurodegeneration marker

proteins (alpha synuclein: F1-2, APP: F3-4) and vascular remodeling - (Apo A-I/E/J: F3-5) related

cargo together with brain derived oxysterols, indicating their specific involvement in the modulation

of vascular diseases. A high content of mitochondrial proteins and enrichment of cardiolipins in F5,

render this fraction a candidate for secreted autophagic vesicles.

Polymorphisms of ATP10D are associated with diabesity and low HDL in mice and man

Alexander Sigruener1, Christian Wolfrum2, Alfred Boettcher1, Thomas Kopf1, Gerhard Liebisch1, Evelyn Orsó1, Gerd Schmitz1

1 Institute of Clinical Chemistry and Laboratory Medicine, Regensburg University Medical Center, D-93053 Regensburg, Germany; 2 Institute of Molecular Systems Biology, ETH Zürich, CH-8093 Zürich, Switzerland

In EUROSPAN GWAS, combining SNPs with lipid species data, disease risk and morbidity,

polymorphic genes in sphingolipid/fatty acid metabolism were significantly associated with

circulating lipid species and diabesity. Two ATP10D gene SNPs correlated significantly with

circulating C16:0 and C24:1 hexosylceramide levels, obesity and insulin resistance. ATP10D is a

P4 type ATPase associated to HDL modulation in mice. C57BL/6 mice express a truncated form of

ATP10D and easily develop obesity and insulin resistance on high-fat diet.


[28]

To get more insight into the physiological function of ATP10D a transgenic mouse model was

created on the background of the commonly used C57/B6J laboratory strain. Plasma lipoprotein

fractions were isolated by Fast Phase Liquid Chromatography. Cholesterol and triglycerides in the

fractions were determined with commercial kits Transcriptomic effects were determined using

mRNA microarrays and RT-PCR. Lipidomic alterations were measured by gas

chromatography/mass spectrometry and by electrospray ionization tandem mass spectrometry.

Compared to ATP10D transgenic, deficient mice gain 20% more weight on high-fat diet, revealed

significantly increased triglycerides levels, decreased O2 consumption/CO2 production,

significantly elevated glucose/insulin levels and reduced insulin sensitivity. A pronounced shift was

observed for lipoprotein-bound triglycerides. SCD1 expression was increased in deficient mice

along with altered SCD1-related fatty acid and lipid species patterns.

In summary we showed that rescue of ATP10D function in mice on C57BL/6 background leads to

reduced HFD induced obesity and insulin resistance, altered hepatic expression of lipid-

metabolism associated genes, including elevated SCD1 expression which attends with increased

SCD1 product/substrate ratios, and distinct changes in the plasma lipoprotein pattern.

Glycerophospholipid and sphingolipid species and mortality: the Ludwigshafen Risk and Cardiovascular Health (LURIC) study

Alexander Sigruener1*, Marcus E. Kleber2, Susanne Heimerl1, Gerhard Liebisch1, Gerd Schmitz1, Winfried Maerz2,3,4

1 Institute for Laboratory Medicine and Transfusion Medicine, Regensburg University Medical Center, Regensburg, Germany; 2 Medical Clinic V, Mannheim Medical Faculty, University of Heidelberg, Mannheim, Germany; 3 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria; 4 Synlab Academy, Synlab Services GmbH, Mannheim, Germany

Vascular and metabolic diseases cause half of total mortality in Europe. New prognostic markers

would provide a valuable tool to improve outcome. First evidence supports the usefulness of

plasma lipid species as easily accessible markers for certain diseases. Here we analyzed

association of plasma lipid species with mortality in the Ludwigshafen Risk and Cardiovascular

Health (LURIC) study.

Plasma lipid species were quantified by electrospray ionization tandem mass spectrometry and

Cox proportional hazards regression was applied to assess their association with total and

cardiovascular mortality.

Overall no differences were detected between total and cardiovascular mortality. Highly

polyunsaturated phosphatidylcholine species together with lysophosphatidylcholine species and

long chain saturated sphingomyelin and ceramide species seem to be associated with a protective

effect. The predominantly circulating phosphatidylcholine-based as well as

phosphatidylethanolamine-based ether species and phosphatidylethanolamine species were

positively associated with total and cardiovascular mortality. Saturated and monounsaturated

phosphatidylcholine species, especially phosphatidylcholine 32:0 (most probably dipalmitoyl-

phosphatidylcholine) and palmitate containing sphingomyelin and ceramide species showed

together with 24:1 containing sphingomyelin and ceramide species strongest positive association

with mortality. A quotient of the sums of the six most protective species and the six species with

the strongest positive mortality association indicated an almost 3-fold increased risk of mortality,

which was higher than the hazard ratio for known risk factors in our cohort.


[29]

Plasma lipid species levels and especially ratios of certain species may be valuable prognostic

marker for cardiovascular and total mortality.

Monocyte to Macrophage Differentiation Goes along with Modulation of the Plasmalogen Pattern through Transcriptional Regulation

Stefan Wallner, Margot Grandl, Tatiana Konovalova, Alexander Sigrüner, Thomas Kopf, Markus Peer, Evelyn Orsó, Gerhard Liebisch, Gerd Schmitz

Institute of Clinical Chemistry and Laboratory Medicine, Regensburg University Medical Center, D-93053 Regensburg, Germany

Background

Dysregulation of monocyte-macrophage differentiation is a hallmark of vascular and metabolic

diseases and associated with persistent low grade inflammation. Plasmalogens represent ether

lipids that play a role in diabesity and previous data show diminished plasmalogen levels in obese

subjects. We therefore analyzed transcriptomic and lipidomic changes during monocyte-

macrophage differentiation in vitro using a bioinformatic approach.

Methods

Elutriated monocytes from 13 healthy donors were differentiated in vitro to macrophages using

rhM-CSF under serum-free conditions. Samples were taken on days 0, 1, 4 and 5 and analyzed for

their lipidomic and transcriptomic profiles.

Results

Gene expression analysis showed strong regulation of lipidome-related transcripts. Enzymes

involved in fatty acid desaturation and elongation were increasingly expressed, peroxisomal and

ER stress related genes were induced. Total plasmalogen levels remained unchanged, while the

PE plasmalogen species pattern became more similar to circulating granulocytes, showing

decreases in PUFA and increases in MUFA. A partial least squares discriminant analysis (PLS/DA)

revealed that PE plasmalogens discriminate the stage of monocyte-derived macrophage

differentiation. Partial correlation analysis could predict novel potential key nodes including

DOCK1, PDK4, GNPTAB and FAM126A that might be involved in regulating lipid and especially

plasmalogen homeostasis during differentiation. An in silico transcription analysis of lipid related

regulation revealed known motifs such as PPAR-gamma and KLF4 as well as novel candidates

such as NFY, RNF96 and Zinc-finger proteins.

Conclusion

Monocyte to macrophage differentiation goes along with profound changes in the lipid-related

transcriptome. This leads to an induction of fatty-acid desaturation and elongation. In their PE-

plasmalogen profile macrophages become more similar to granulocytes than monocytes, indicating

terminal phagocytic differentiation. Therefore PE plasmalogens may represent potential biomarkers

for cell activation. For the underlying transcriptional network we were able to predict a range of

novel central key nodes and underlying transcription factors using a bioinformatic approach.


[30]

Lipidomic and transcriptomic profiling of human macrophages and fibroblasts in

Niemann-Pick type C disease: implications to pathogenesis

Evelyn Orsó1, Alexander Sigrüner1, Gerhard Liebisch1, Hans H. Klünemann2, Thomas Kolter3 & Gerd Schmitz1

1 Institute for Laboratory Medicine and Transfusion Medicine, University Hospital Regensburg, Regensburg, Germany

2 Department of Psychiatry, University of Regensburg, Regensburg, Germany

3 LiMES Program Unit Membrane Biology & Lipid Biochemistry, University of Bonn, Bonn, German

Niemann-Pick type C disease (NPC) is a lipid traffic disorder, characterized by endosomal

accumulation of cholesterol and other lipids due to loss-of-function mutations in NPC1 (or

NPC2) genes.

In order to characterize cellular pathways contributing to intracellular lipid traffic, control

and NPC1 mutant macrophages and fibroblasts were compared. Macrophages were

challenged either by endolysosomal phospholipidosis induction (i.e. loading with oxidized

LDL /oxLDL/) or by lipid droplet storage induction (i.e. by enzymatically modified LDL

/eLDL/). Fibroblasts were subjected to fetal calf serum (FCS) in order to promote

lipoprotein uptake. Transcriptional profiling and apoptosis assays were performed parallel

with analysis of molecular lipid species. Particular attention was turned to organelle

signature lipids, e.g. cardiolipin (CL) for mitochondria, bis(monoacylglycero)phosphate

(BMP) for endolysosomes.

The well-developed endolysosmal system of macrophages was able to degrade and traffic

lipids even in NPC1 deficiency, since BMP, as biomarker for endolysosomal dysfunction

and phospholipidosis, did not show major accumulation in NPC cells. Cellular lipid loading

was paralleled with increased mitochondrial activity and enhanced CL levels, which was

altered in NPC macrophages. By contrast, NPC fibroblasts, showing limited

endolysosomal/mitochondrial capacities, exerted phospholipidosis with BMP accumulation,

increased contents of C22 and C24 sphingomyelin species, mitochondrial dysfunction and

apoptosis susceptibility.

Acknowledgements: The project was supported by the European Community's Seventh

Framework Programme (FP7/2007–2013) under grant agreement n° 202272, IP-Project

LipidomicNet.

Adaptation of human macrophages to HDL-mediated cellular stress: transcriptional

regulation and selective uptake of HDL-lipids

Evelyn Orsó1, Alexander Sigrüner1, Gerhard Liebisch1, Tatiana Konovalova1 & Gerd Schmitz1

1 Institute for Laboratory Medicine and Transfusion Medicine, University Hospital Regensburg, Regensburg, Germany

The association of plasma high density lipoprotein (HDL)-cholesterol (HDL-C) with reduced risk for

cardiovascular disease (CVD) is well described. In addition to cholesterol efflux and reverse

cholesterol transport, HDL and particularly the HDL3–subclass exerts pleiotropic effects in diverse

cells.

The aim of the present study was to characterize the effect of HDL3–stress on the transcriptome

and lipidome of monocyte-derived macrophages and foam cells.


[31]

Human monocyte-derived macrophages and lipid-loaded (either with enzymatically-modified low

density lipoprotein (eLDL) or with oxidized LDL (oxLDL)) cells were treated with HDL3.

Transcriptomic and lipidomic profiling was carried out by microarrays and by mass spectrometry,

respectively.

In vitro HDL3–stress induced multiple gene regulations: (i) SREBP-dependent activation of nearly

all genes in cholesterol/fatty acid synthesis and sterol uptake, (ii) up-regulation of vesicular lipid

traffic, (iii) down-regulation of sterol/phospholipid efflux. In addition, novel HDL3-responsive genes

(C14orf1, MMAB) were also found, likely involved in cholesterol traffic.

In addition to cholesterol efflux, a selective uptake of cholesterylesters from HDL3 was also

observed. It was most prominent in unloaded cells, marginal in oxLDL-loaded, and absent in eLDL-

loaded cells. Consistent with the parallel regulation of sphingomyelin (SPM) and cholesterol,

cellular SPM18:0 and SPM16:1 content was also increased in unloaded, but not foam cells. In

addition, cellular adaptation to HDL3 stress led to the up-regulation of phosphatidylcholine and

phosphatidylethanolamine, but not phosphatidylserine, ceramide (Cer) and

polyglycerophospholipid (pGP) species, irrespective of lipid-load.

Taken together, HDL3 is a significant modulator of cellular cholesterol and glycerophospholipids,

without major influence on pGP and Cer. The adaptation of macrophages in response to HDL3 may

contribute to the reduction of CVD risk.

Acknowledgements: The project was supported by the European Community's Seventh

Framework Programme (FP7/2007–2013) under grant agreement n° 202272, IP-Project

LipidomicNet.

Valid serum creatinine measurement in face of severe icteric interference for reliable MELD Scoring

C. Gnewuch*, S. Reichl, G. Schmitz

University Hospital Regensburg, Institute for Clinical Chemistry and Laboratory Medicine, Regensburg, Germany

Objectives: Invalid serum creatinine owing to extreme bilirubin concentrations is a major problem

in MELD Score calculation for liver transplant allocation. Even with the less susceptible enzymatic

measurement procedures falsely reduced creatinine levels are possible. We evaluated if a simple

additional dilution step prior to enzymatic reaction in the clinical chemistry autoanalyzer sufficiently

eliminates icteric interference yielding valid creatinine values.

Methods: Serum Creatinine, total bilirubin and icteric index of 51 serial two-month cases with

creatinine values, that were commented as invalid in the lab report due to severe icteric

interference, were re-measured on a Siemens Dimension Vista® 1500 analyzer after manual 1:2

dilution with 0.9% NaCl.

Results: In all but two cases sample dilution yielded bilirubin concentrations that fell below the

critical threshold of 20 mg/dL where serum creatinine measurements become invalid. Pre-diluted

creatinine values (CV < 6%) were higher than the invalid original values at an average of 16% with

an optimal linear correlation (r = 0,9969).

Conclusion: More than 90% of invalid creatinine measurements in severe icteric samples can

easily be prevented by a moderate 1:2 pre-dilution step. This leads to more reliable MELD Score

calculation and in most cases to a higher priority in liver transplant allocation.


[32]

Lipidomic analysis of human blood cells – lessons from platelet lipid species alterations during aging

Katharina Ruebsaamen, Gerhard Liebisch, Alfred Boettcher, Gerd Schmitz

Institute of Clinical Chemistry and Laboratory Medicine, Regensburg University Medical Center, D-93053 Regensburg, Germany

Circulating blood cell lipid composition may become increasingly important to provide new insights

into cellular lipid abnormalities in diseases such as atherosclerosis and thrombosis. For this reason

the lipid species pattern in monocytes, lymphocytes, granulocytes, platelets and red blood cells of

healthy volunteers were compared using electrospray ionization tandem mass spectrometry (ESI-

MS/MS). Among the examined blood cells striking differences were detected, e.g. the ceramide

levels in granulocytes (2.6 mol%) were five-fold and the proportion of cholesteryl esters (CE) in

platelets (2.5 mol%) was at least four-fold higher than in other analyzed blood cells. Moreover, the

CE lipid species pattern of platelets and plasma showed a nearly identical distribution. Therefore

we also analyzed by ESI-MS/MS the plasma and platelet lipids of 50 platelet apheresis

concentrates stored for five days at 22°C. Distinct lipid shifts (for phosphatidylserine and

lysophosphatidylcholine) and interactions (for phosphatidylcholine, free cholesterol and CE) could

be detected during platelet concentrate storage. In this context the role of lipids could contribute to

a better understanding of cellular senescence and platelet storage leading to novel therapeutic

targets for the control of thrombosis and platelet-mediated diseases. In conclusion, the current

studies provide a detailed comparison of lipid species in circulating blood cells of healthy human

donors as well as in platelet concentrates used for transfusion purposes. This work could serve as

a reference for studies in different patient cohorts directed towards discovery of novel lipid

biomarkers.

Loading of primary monocyte derived macrophages with modified lipoproteins differentially induces saturation specific alterations in the plasmalogen profile

Stefan Wallner, Evelyn Orsó, Alexander Sigrüner, Margot Grandl, Tatiana Konovalova, Markus Peer, Gerhard Liebisch, and Gerd Schmitz

Institute for Laboratory Medicine and Transfusion Medicine, Regensburg University Medical Center, Germany

Background

Plasmalogens are glycerol-phospholipids that contain a vinyl ether moiety bound in the sn-1-

position. A range of functions has been proposed for them: (1) storage of precursor molecules for

inflammatory mediators, (2) modulation of membrane fluidity, (3) anti-oxidative properties and (4)

modulation of cholesterol efflux. This makes them candidates for playing a role in macrophages

under conditions of lipid stress. Especially atherogenically modified LDL is preferentially taken up

by macrophages. Enzymatically degraded LDL particles (eLDL) leads to the formation of foam cells

and the reaction of LDL with free radicals induces the formation of oxidatively modified LDL

(oxLDL). oxLDL is more reactive than native LDL or eLDL and leads to tissue damage. The aim of

this study was to better understand the effect of modified lipoproteins on macrophages, especially

the effect on plasmalogen species has not been studied before.

Methods

Elutriated monocytes from nine healthy donors were differentiated in vitro to macrophages for four

days using rhM-CSF under serum-free conditions. Macrophages were loaded with LDL-species for

24 hours followed by harvesting. In addition to native low-density lipoproteins (LDL, 40 µg/ml),


[33]

enzymatically modified LDL (eLDL, 40 µg/ml) and oxidized LDL (oxLDL, 80 µg/ml) was used. Cells

were then lysed and lipid mass spectrometry and gene array analysis performed. Data was

subjected to partial correlation analysis and clustered using the OH-PIN algorithm in Cytoscape.

BinGO GeneOntology (GO) analysis was used to characterize the resulting clusters.

Results

Total plasmalogen levels did not change significantly after loading with modified lipoproteins.

Instead changes in plasmalogen levels were highly saturation specific and did not directly reflect

the species pattern that was found in the lipoproteins used for loading. In eLDL treated cells,

plasmalogen species containing saturated, mono-unsaturated and three-times unsaturated acyl-

residues in sn-2 position were lower than in controls. In contrast treatment with oxLDL led to a

specific reduction in plasmalogens containing saturated acyl residues despite the cells being

loaded strongly with these species. Furthermore adipokine production was differentially regulated

on the transcriptomic level. Transcripts for adipokines in macrophages were generally induced or

unchanged by treatment with both modified lipoproteins. No adipokine was significantly

downregulated. This effect was independent of whether an adipokine was primarily pro- or anti-

inflammatory. Leptin, apelin and omentin were induced by both treatments, while RBP-4

upregulation was specific for eLDL treated cells and induction of visfatin was specific for oxLDL

treated cells. Additionally Notch signaling was modulated specifically by oxLDL. Partial correlation

analysis of gene transcripts and plasmalogen species revealed potential hubs in the transcriptional

network. The resulting network was subjected to clustering analysis and resulting sub-networks

were analyzed for overrepresentation of GO-categories. The most strongly interconnected region

in eLDL treated cells was associated with sterol metabolism. Overloading with hydrolyzed fatty acid

chains from LDL particles resulted in a general downregulation including key transcripts such as

the LDL-receptor, HMG-Co-A-reductase and fatty acid desaturases FADS1 and FADS2.

Plasmalogens formed a cluster that was connected to apoptosis related genes. In oxLDL treated

cells, major GO categories included apoptosis, stress and defense response. Similarly to loading

with eLDL, sterol metabolism was also affected by oxLDL but to a lesser extent. The gene-cluster

containing the most densely connected transcript network was classified by GO analysis to be

related to cell cycle regulation.

Conclusion

Modified lipoproteins are taken up by in vitro differentiated macrophages and induce specific

effects on the transcriptome and on the plasmalogen species pattern. eLDL leads to foam cell

formation with storage of lipids in large intracellular lipid droplets. OxLDL in contrast induces a

phospholipidosis phenotype. While total plasmalogen levels remained constant for both treatments

the lipoproteins induced saturation specific effects on the cellular plasmalogen profile.


[34]

Organizing Committee

The organizing committee is comprised of researchers and manager from the Institute for

Laboratory Medicine and Transfusion Medicine at University Hospital Regensburg:

Dr. Evelyn Orsó, Dr. Gerhard Liebisch, Dr. Susanne Heimerl & Dr. Megi Sharikadze

Institute for Laboratory Medicine and Transfusion Medicine at University Hospital Regensburg

http://www.ukr.de/kliniken-institute/klinische-chemie/index.php

Meeting Venues

Conference venue

Lecture Hall A2 (Entrance/Eingang “West”), University Hospital Regensburg (UHR). See the

enclosed UHR map on the last page (available in German only). The large red arrow shows the

main entrance (Haupteingang), the light blue circle indicates the venue in the part A2 of the

hospital (on the right side of the map). The end-stop of Bus No.6 is in front of D1.

The Institute for Laboratory Medicine and Transfusion Medicine is located in C3 part of the

University Hospital Regensburg.

Transport hub-Albertstraße (satellite view). Marked: main train station (with a red circle), bus-stop

“Albertstraße” for Bus No.6 direction “Klinikum” (with a red star) and other bus-stops (along red line

Albertstraße,).

http://www.ukr.de/kliniken-institute/klinische-chemie/index.php


[35]

Directions to the venue

By car via the motorway A3: (Nuremberg-Passau / Passau-Nuremberg)

Take the exit "Universität / Klinikum Regensburg" (University / University Hospital Regensburg).

Follow the signs to ‘Klinikum (University Hospital Regensburg)’ by turning off at the next

intersection. After a few hundred meters, the car park of the University Hospital is on your left.

By car via the motorway A93: (Munich-Weiden / Weiden-Munich)

At the motorway junction Regensburg, follow the motorway A3 to Passau. Take exit ‘Universität /

Klinikum Regensburg (University / University Hospital Regensburg)’. Follow the signs to ‘Klinikum

(University Hospital Regensburg)’ by turning off at the next intersection. After a few hundred

meters, the car park of the University Hospital is on your left.

By bus and train

Buses no. 6 and 19 connect the University Hospital with the central railway station, which is close

to the central bus station in Albert Street (Ablertstraße). From there, buses no. 6 (Klinikum-

University Hospital) and 19 (Lengfeld/Bad Abbach) take about 10 min. to get to the University

Hospital.

Please note that bus no. 6 leaves just around the corner of Albert Street at Galgenberg Bridge.


[36]

Directions to hotels

All hotels suggested by a cooperating travel agency are located within the city center not far from

bus No.6 route which goes to University Hospital Regensburg (direction "Klinikum", the last stop).

Most of the hotels are located within the city center not far from bus No.6 route which goes directly

to University Hospital Regensburg (direction "Klinikum"). From bus-stop "Albertstraße" you can

change from different bus-lines to the bus-line 6, direction “Klinikum”. "Albertstraße" is the main

hub of the local bus network with multiple bus stops located not far away from the main train

station. Conveniently, city center/old town is located on walking distance from the main train

station.

Weblinks

European commission http://ec.europa.eu/index_en.htm

Seventh Framework Programme (FP7) http://cordis.europa.eu/fp7/find-doc_en.html

UNESCO World Heritage Site Regensburg http://www.regensburg.de/tourismus/de/33377

Regensburg-World Heritage Visitor Centre http://www.regensburg.de/tourismus/de/57972

LipidomicNet final meeting related information you can find also online

http://www.lipidomicnet.org/index.php/LipidomicNet_Final_Meeting_-_10/2012

Our supporters

German Society for Fat Science http://www.dgfett.de

German Society for Transfusion Medicine and Immunohematology http://www.dgti.de

German Association for Clinical Chemistry and Laboratory Medicine (DGKL) http://www.dgkl.de

European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) http://www.efcclm.eu

European FP7 Health IP-Project LipidomicNet (GA 202272) http://www.lipidomicnet.org

European Society of Pharmacogenetics and Theranostics (ESPT) www.esptnet.eu

Hungarian Biochemical Society http://www.mbkegy.hu

Hungarian Society of Laboratory Medicine http://www.mldt.hu

International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) http://www.ifcc.org

The German Society of Proteome Research http://www.dgpf.org

University Hospital Regensburg http://www.ukr.de

http://ec.europa.eu/index_en.htm

http://cordis.europa.eu/fp7/find-doc_en.html

http://www.regensburg.de/tourismus/de/33377

http://www.regensburg.de/tourismus/de/57972

http://www.lipidomicnet.org/index.php/LipidomicNet_Final_Meeting_-_10/2012

http://www.dgfett.de/

http://www.dgti.de/

http://www.dgkl.de/

http://www.efcclm.eu/

http://www.lipidomicnet.org/

http://www.esptnet.eu/

http://www.mbkegy.hu/

http://www.mldt.hu/

http://www.ifcc.org/

http://www.dgpf.org/

http://www.ukr.de/


[37]

Conference dinner

Conference dinner will take place at Herzogssaal. www.herzogssaal.com

Domplatz 3, 93047 Regensburg

Phone +49 (0)941 / 586 146 - 10

Fax +49 (0)941 / 586 146 – 29

E-Mail: [email protected]

http://www.herzogssaal.com/

mailto:[email protected]


[38]

Sponsors

We would like to acknowledge the generous contributions of our sponsors (sorted alphabetically).

AB SCIEX Germany GmbH

http://www.absciex.com

Agilent Technologies Sales & Services GmbH & Co. KG

http://www.agilent.com

Avanti Polar Lipids, Inc.

http://www.avantilipids.com

Becton Dickinson GmbH

http://www.bd.com

BIOZOL Diagnostica Vertrieb GmbH

http://www.biozol.de

FluidX Ltd.

http://www.fluidx.eu

Fresenius Fresenius SE & Co. KGaa

http://www.fresenius.de

Immucor Inc.

http://www.immucor.com

Siemens AG. (The main sponsor)

http://www.siemens.com

Sysmex Europe GmbH

http://www.sysmex-europe.com

R&D Systems, Inc.

http://www.rndsystems.com

Roche Diagnostics Deutschland GmbH

http://www.roche.com

Sarstedt Ag. & Co.

http://www.http://sarstedt.com

Thermo Fisher Scientific Inc.

http://www.thermoscientific.com

Waters Corporation GmbH

http://http://www.waters.com

http://www.absciex.com/

http://www.agilent.com/

http://www.avantilipids.com/

http://www.bd.com/

http://www.biozol.de/

http://www.fluidx.eu/

http://www.fresenius.de/

http://www.immucor.com/

http://www.siemens.com/

http://www.sysmex-europe.com/

http://www.rndsystems.com/

http://www.roche.com/

http://www.http/sarstedt.com

http://www.thermoscientific.com/

http://http/www.waters.com


[39]

Sponsors

KURSRÄUME1 - 3 / 1. OG

PERSONALCASINO1. UG

B 3

B 2

B 1

Z

A

H 1

B 4

D 1 D 2

C 1 C 2 C 3 C 4

C 5

A 2

D 3

D 4

H 5

JCC

EG

2. UG

(11) KIEFERORTHOPÄDIE(14) MUND-, KIEFER-, GESICHTSCHIR.

(24) ZAHNÄRZTL. PROTHETIK(25) ZAHNERHALTUNG, PARODONTOLOGIE

(3) CHIRURGIE / (23) UNFALLCHIR.

(8) INNERE MED. I

(8) INNERE MED. I(10) INNERE MED. III

(9) INNERE MED. II(LUNGE, NIERE)

Stat. 15 Stat. 14

Stat. 11 Stat. 101.

OG

EG

INTENSIV-STATIONEN

92 u. 93

(21) STRAHLEN-THERAPIE

NMR

(20) RÖNTGEN-DIAGNOSTIK1.

UG

Stat. 13

Stat. 12

(17) NUKLEAR-MEDIZIN

INTENSIV-STATION

91

NOTAUF-NAHME

2. UG

Stat. 20

Stat. 18Stat. 16DIALYSE

Stat. 21

Stat. 19Stat. 17

1. UG

2. UG

3. UG

KAPELLE

PATIENTEN-AUFNAHME

INFOTHEK

HAUPT-EINGANG

(9) INNERE MED.II(HERZ)

(19) PSYCHO-SOMATIK

Stat. 49

Stat. 48

Stat. 53 Stat. 57 Stat. 59

Stat. 52 Stat. 56 Stat. 58

Stat. 46

Stat. 47

Stat. 51

Stat. 50

Stat. 55

Stat. 54

Stat. 61

Stat. 60

2. OG

1. OG

EG

1. UG

INTENSIV-STATION

90

(2) AUGENHEILKUNDE(15) NEUROCHIRURGIE

(4) DERMATOLOGIE(5) HNO - HEILKUNDE

(7) ZENTRALABOR (6) HERZ - THORAX - CHIRURGIE(1) ANÄSTHESIE / SCHMERZAMBULANZ

(22) THORAXCHIRURGIE

INTENSIV-STATION

97

(7) TRANSFUSIONS-MEDIZIN

PHYSIOTHERAPIE

PATHOLOGIEMIKRO-

BIOLOGIE

(16) NEURO-PATHOLOGIE

Stat. 85

1. OG

EG

1. UG

Stat. 83

81

KLINIKUM Linien 6 / 19

KLINIKUM WEST Linien 6 / 6S

H 2

H 3

H 4

HUMANGENETIK

1. OG

2. OG

EG

1. UG

EPIDEMIOLOGIE

IMMUNOLOGIE

(7) KLINISCHE CHEMIE

APOTHEKE

HYGIENE

DEKANAT

VKKK ELTERNHAUS

GROSSERHÖRSAAL

1. OG

KLEINERHÖRSAAL

1. OG

HÖRSAAL A2 - EG

HÖRSAALPATHOLOGIE

HÖRSAALZAHNKLINIKEN

1. OG

ELEKTRO-TECHNIK

KFA

MEDIZIN-TECHNIK

StATUR

Ba Bank /GeldautomatIm Bauteil A/EG

Br Briefkasten & MarkenIm Bauteil A/EG

C CafeteriaIm Bauteil A / EG und A2/EG

GetränkeautomatenIm Bauteil A / EG; B1/1UG; B3/2UG; C5/EG

G

Ko KopfhörerIm Bauteil A/EG; C5/EG

KioskIm Bauteil A/EG Ki

SpielmöglichkeitenIm Bauteil B2/1UG; C2/EG

TV & Telefonkarten-automatenBauteil A/EG; C5/EG

Wickel- & StillraumIm Bauteil B1/EG

S

TV

W

Treppen

Ba

Br

CCG

G

G

G

Ko

Ki

S

STV

W

(12) KINDER- UND JUGENDMEDIZIN

(KUNO)

EINGANGWEST

Ambulanz und Tagesklinik

V 1

Stat. 84

INTENSIVSTATION

Stat. 82

(13)

(18) PÄD. HÄMATOLOGIE, ONKOLOGIE UND

STAMMZELLTRANSPLANTATION

TV

TV

Wegweiser Universitätsklinikum Regensburg

Universitätsklinikum Regensburg (UKR): Franz-Josef-Strauß-Allee 11 Telefon: 0941 944-0 93053 Regensburg Internet: www.ukr.de

Leitstelle der Notaufnahme Telefon: 0941 944-2310 Ärztliche Leitung PD Dr. Markus Zimmermann

Herausgeber: Universitätsklinikum Regensburg/ UnternehmenskommunikationRedaktion und Fotos: UKRJanuar 2014

international conference healthcare integrated biobanking ... · 2014 conference program version...

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