Publications by authors named "Kristaps Klavins"

30 Publications

  • Page 1 of 1

Metabolome and lipidome derangements during a severe mast cell activation event in a patient with indolent systemic mastocytosis.

J Allergy Clin Immunol 2021 Apr 14. Epub 2021 Apr 14.

Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.

Background: The number of mast cells in various organs is manifold elevated in individuals with systemic mastocytosis. Degranulation can lead to life-threatening symptomatology. No data have been published about the alterations of the metabolome and lipidome during an attack.

Objective: To analyze metabolomics and lipidomics changes during the acute phase of a severe mast cell activation event.

Methods: Forty-three metabolites and 11 lipid classes comprising 200 subvariants from multiple plasma samples in duplicate, covering 72 hours of a severe mast cell activation attack with nausea and vomiting, were compared to two baseline samples using quantitative liquid chromatography mass spectrometry.

Results: A strong enterocyte dysfunction reflected in an almost 20-fold reduction in the functional small bowel length extrapolated from strongly reduced ornithine and citrulline concentrations was very likely secondary to severe endothelial cell dysfunction with hypoperfusion and extensive vascular leakage. Highly increased histamine and lactate concentrations accompanied the peak in clinical symptoms. Elevated asymmetric and symmetric dimethylarginine combined with reduced arginine levels compromised endothelial nitric oxide synthase activity and nitric oxide signaling. Specific and extensive depletion of many lysophosphatidylcholine variants indicates localized autotaxin activation and lysophosphatidic acid release. A strong correlation of clinical parameters with histamine concentrations and symptom reduction after 100-fold elevated plasma diamine oxidase concentrations imply histamine is the key driver of the acute phase.

Conclusions: Rapid elimination of elevated histamine concentrations using recombinant human diamine oxidase, supplementation of lysophosphatidylcholine for immunomodulation, inhibition of autotaxin activity and/or blockade of lysophosphatidic acid receptors might represent new treatment options for life-threatening mast cell activation events.
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http://dx.doi.org/10.1016/j.jaci.2021.03.043DOI Listing
April 2021

The PI3K pathway preserves metabolic health through MARCO-dependent lipid uptake by adipose tissue macrophages.

Nat Metab 2020 12 16;2(12):1427-1442. Epub 2020 Nov 16.

Institute for Vascular Biology, Center for Physiology and Pharmacology, Medical University Vienna, Vienna, Austria.

Adipose tissue macrophages (ATMs) display tremendous heterogeneity depending on signals in their local microenvironment and contribute to the pathogenesis of obesity. The phosphoinositide 3-kinase (PI3K) signalling pathway, antagonized by the phosphatase and tensin homologue (PTEN), is important for metabolic responses to obesity. We hypothesized that fluctuations in macrophage-intrinsic PI3K activity via PTEN could alter the trajectory of metabolic disease by driving distinct ATM populations. Using mice harbouring macrophage-specific PTEN deletion or bone marrow chimeras carrying additional PTEN copies, we demonstrate that sustained PI3K activity in macrophages preserves metabolic health in obesity by preventing lipotoxicity. Myeloid PI3K signalling promotes a beneficial ATM population characterized by lipid uptake, catabolism and high expression of the scavenger macrophage receptor with collagenous structure (MARCO). Dual MARCO and myeloid PTEN deficiencies prevent the generation of lipid-buffering ATMs, reversing the beneficial actions of elevated myeloid PI3K activity in metabolic disease. Thus, macrophage-intrinsic PI3K signalling boosts metabolic health by driving ATM programmes associated with MARCO-dependent lipid uptake.
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http://dx.doi.org/10.1038/s42255-020-00311-5DOI Listing
December 2020

Hepatocyte-intrinsic type I interferon signaling reprograms metabolism and reveals a novel compensatory mechanism of the tryptophan-kynurenine pathway in viral hepatitis.

PLoS Pathog 2020 10 12;16(10):e1008973. Epub 2020 Oct 12.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse, Vienna, Austria.

The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis.
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http://dx.doi.org/10.1371/journal.ppat.1008973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580883PMC
October 2020

Alterations of Lipid Metabolism With Age and Weight in Companion Dogs.

J Gerontol A Biol Sci Med Sci 2021 Feb;76(3):400-405

Department of Biology, University of Alabama at Birmingham.

The companion dog has recently been promoted as powerful translational model of aging. However, while dogs share environments with their human owners and develop many of the same age-related morbidities, little is known about the underlying mechanisms that drive their health and longevity. In addition, dogs have a well described phenotypic pattern in which small dogs live significantly longer than large dogs, such that weight can be used as a crude proxy for longevity. To investigate this pattern, we completed a small lipidomics study on 41 dogs in the Birmingham, Alabama, United States, area to determine individual circulating lipids that were associated with age and body weight. We discovered that sphingomyelins were significantly higher in large, short-lived dogs, independent of age, and triglycerides were higher in older dogs of all sizes. Our results point towards physiological differences that may explain a portion of the variation in longevity seen in companion dogs.
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http://dx.doi.org/10.1093/gerona/glaa186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907484PMC
February 2021

Imaging of metabolic activity adaptations to UV stress, drugs and differentiation at cellular resolution in skin and skin equivalents - Implications for oxidative UV damage.

Redox Biol 2020 10 19;37:101583. Epub 2020 Jul 19.

Department of Dermatology Medical University of Vienna, Austria; Christian Doppler Laboratory for Biotechnology of Skin Aging, Austria. Electronic address:

The epidermis is a multi-layered epithelium that consists mainly of keratinocytes which proliferate in its basal layer and then differentiate to form the stratum corneum, the skin's ultimate barrier to the environment. During differentiation keratinocyte function, chemical composition, physical properties, metabolism and secretion are profoundly changed. Extrinsic or intrinsic stressors, like ultraviolet (UV) radiation thus may differently affect the epidermal keratinocytes, depending on differentiation stage. Exposure to UV elicits the DNA damage responses, activation of pathways which detoxify or repair damage or induction of programmed cell death when the damage was irreparable. Recently, rapid diversion of glucose flux into the pentose phosphate pathway (PPP) was discovered as additional mechanism by which cells rapidly generate reduction equivalents and precursors for nucleotides - both being in demand after UV damage. There is however little known about the correlation of such metabolic activity with differentiation state, cell damage and tissue localization of epidermal cells. We developed a method to correlate the activity of G6PD, the first and rate-limiting enzyme of this metabolic UV response, at cellular resolution to cell type, differentiation state, and cell damage in human skin and in organotypic reconstructed epidermis. We thereby could verify rapid activation of G6PD as an immediate UVB response not only in basal but also in differentiating epidermal keratinocytes and found increased activity in cells which initiated DNA damage responses. When keratinocytes had been UVB irradiated before organotypic culture, their distribution within the skin equivalent was abnormal and the G6PD activity was reduced compared to neighboring cells. Finally, we found that the anti-diabetic and potential anti-aging drug metformin strongly induced G6PD activity throughout reconstructed epidermis. Activation of the protective pentose phosphate pathway may be useful to enhance the skin's antioxidant defense systems and DNA damage repair capacity on demand.
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http://dx.doi.org/10.1016/j.redox.2020.101583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767734PMC
October 2020

Simple, sensitive and specific quantification of diamine oxidase activity in complex matrices using newly discovered fluorophores derived from natural substrates.

Inflamm Res 2020 Sep 1;69(9):937-950. Epub 2020 Jun 1.

Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090, Vienna, Austria.

Objective: To measure diamine oxidase (DAO) activity with high sensitivity in complex matrices like plasma or tissue extracts radioactive putrescine or horseradish peroxidase (HRP)/hydrogen peroxide (HO) coupling must be used. The use of radioactive material should be avoided and HRP/HO coupling is compromised by antioxidants.

Methods And Results: Condensation of ortho-aminobenzaldehyde (oABA) with delta-1-pyrroline and delta-1-piperideine, the autocyclization products of the DAO-oxidized natural substrates putrescine and cadaverine, generates new quinazoline fluorophores with absorption and excitation maxima of 430 and 460 nm, respectively, and peak emission at 620 nm. Fluorescent-based detection limits are 20-40 times lower compared to absorption measurements. This assay can be used to measure DAO activity in human plasma after spiking recombinant human (rh)DAO, in rat plasma after intravenous rhDAO administration, in pregnancy plasma and in tissue extracts of DAO wild-type and knock-out mice. Using rat plasma the correlation between rhDAO activity and ELISA data is 99%. Human and rat plasma without DAO spiking and tissue extracts from DAO knock-out mice showed stable and low fluorescence in the presence of high substrate concentrations.

Conclusions: Incubation of DAO with the natural substrates putrescine and cadaverine and oABA generates novel fluorophores increasing the detection limit compared to absorption measurements at least tenfold. This simple, sensitive and specific assay allows the non-radioactive quantification of DAO activity in complex matrices like plasma and tissue extracts without interference by antioxidants.
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http://dx.doi.org/10.1007/s00011-020-01359-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394931PMC
September 2020

The RESOLUTE consortium: unlocking SLC transporters for drug discovery.

Authors:
Giulio Superti-Furga Daniel Lackner Tabea Wiedmer Alvaro Ingles-Prieto Barbara Barbosa Enrico Girardi Ulrich Goldmann Bettina Gürtl Kristaps Klavins Christoph Klimek Sabrina Lindinger Eva Liñeiro-Retes André C Müller Svenja Onstein Gregor Redinger Daniela Reil Vitaly Sedlyarov Gernot Wolf Matthew Crawford Robert Everley David Hepworth Shenping Liu Stephen Noell Mary Piotrowski Robert Stanton Hui Zhang Salvatore Corallino Andrea Faedo Maria Insidioso Giovanna Maresca Loredana Redaelli Francesca Sassone Lia Scarabottolo Michela Stucchi Paola Tarroni Sara Tremolada Helena Batoulis Andreas Becker Eckhard Bender Yung-Ning Chang Alexander Ehrmann Anke Müller-Fahrnow Vera Pütter Diana Zindel Bradford Hamilton Martin Lenter Diana Santacruz Coralie Viollet Charles Whitehurst Kai Johnsson Philipp Leippe Birgit Baumgarten Lena Chang Yvonne Ibig Martin Pfeifer Jürgen Reinhardt Julian Schönbett Paul Selzer Klaus Seuwen Charles Bettembourg Bruno Biton Jörg Czech Hélène de Foucauld Michel Didier Thomas Licher Vincent Mikol Antje Pommereau Frédéric Puech Veeranagouda Yaligara Aled Edwards Brandon J Bongers Laura H Heitman Ad P IJzerman Huub J Sijben Gerard J P van Westen Justine Grixti Douglas B Kell Farah Mughal Neil Swainston Marina Wright-Muelas Tina Bohstedt Nicola Burgess-Brown Liz Carpenter Katharina Dürr Jesper Hansen Andreea Scacioc Giulia Banci Claire Colas Daniela Digles Gerhard Ecker Barbara Füzi Viktoria Gamsjäger Melanie Grandits Riccardo Martini Florentina Troger Patrick Altermatt Cédric Doucerain Franz Dürrenberger Vania Manolova Anna-Lena Steck Hanna Sundström Maria Wilhelm Claire M Steppan

Nat Rev Drug Discov 2020 07;19(7):429-430

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http://dx.doi.org/10.1038/d41573-020-00056-6DOI Listing
July 2020

A widespread role for SLC transmembrane transporters in resistance to cytotoxic drugs.

Nat Chem Biol 2020 04 9;16(4):469-478. Epub 2020 Mar 9.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.
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http://dx.doi.org/10.1038/s41589-020-0483-3DOI Listing
April 2020

Increased ATP synthesis might counteract hepatic lipid accumulation in acromegaly.

JCI Insight 2020 03 12;5(5). Epub 2020 Mar 12.

Division of Endocrinology and Metabolism, Department of Medicine III, and.

Patients with active acromegaly (ACRO) exhibit low hepatocellular lipids (HCL), despite pronounced insulin resistance (IR). This contrasts the strong association of IR with nonalcoholic fatty liver disease in the general population. Since low HCL levels in ACRO might be caused by changes in oxidative substrate metabolism, we investigated mitochondrial activity and plasma metabolomics/lipidomics in active ACRO. Fifteen subjects with ACRO and seventeen healthy controls, matched for age, BMI, sex, and body composition, underwent 31P/1H-7-T MR spectroscopy of the liver and skeletal muscle as well as plasma metabolomic profiling and an oral glucose tolerance test. Subjects with ACRO showed significantly lower HCL levels, but the ATP synthesis rate was significantly increased compared with that in controls. Furthermore, a decreased ratio of unsaturated-to-saturated intrahepatocellular fatty acids was found in subjects with ACRO. Within assessed plasma lipids, lipidomics, and metabolomics, decreased carnitine species also indicated increased mitochondrial activity. We therefore concluded that excess of growth hormone (GH) in humans counteracts HCL accumulation by increased hepatic ATP synthesis. This was accompanied by a decreased ratio of unsaturated-to-saturated lipids in hepatocytes and by a metabolomic profile, reflecting the increase in mitochondrial activity. Thus, these findings help to better understanding of GH-regulated antisteatotic pathways and provide a better insight into potentially novel therapeutic targets for treating NAFLD.
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http://dx.doi.org/10.1172/jci.insight.134638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141383PMC
March 2020

Environmental arginine controls multinuclear giant cell metabolism and formation.

Nat Commun 2020 01 22;11(1):431. Epub 2020 Jan 22.

Institute for Vascular Biology, Centre for Physiology and Pharmacology, Medical University Vienna, 1090, Vienna, Austria.

Multinucleated giant cells (MGCs) are implicated in many diseases including schistosomiasis, sarcoidosis and arthritis. MGC generation is energy intensive to enforce membrane fusion and cytoplasmic expansion. Using receptor activator of nuclear factor kappa-Β ligand (RANKL) induced osteoclastogenesis to model MGC formation, here we report RANKL cellular programming requires extracellular arginine. Systemic arginine restriction improves outcome in multiple murine arthritis models and its removal induces preosteoclast metabolic quiescence, associated with impaired tricarboxylic acid (TCA) cycle function and metabolite induction. Effects of arginine deprivation on osteoclastogenesis are independent of mTORC1 activity or global transcriptional and translational inhibition. Arginine scarcity also dampens generation of IL-4 induced MGCs. Strikingly, in extracellular arginine absence, both cell types display flexibility as their formation can be restored with select arginine precursors. These data establish how environmental amino acids control the metabolic fate of polykaryons and suggest metabolic ways to manipulate MGC-associated pathologies and bone remodelling.
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http://dx.doi.org/10.1038/s41467-020-14285-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976629PMC
January 2020

Condensation of delta-1-piperideine-6-carboxylate with ortho-aminobenzaldehyde allows its simple, fast, and inexpensive quantification in the urine of patients with antiquitin deficiency.

J Inherit Metab Dis 2020 07 29;43(4):891-900. Epub 2020 Jan 29.

Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria.

Antiquitin (ATQ) deficiency leads to tissue, plasma, and urinary accumulation of alpha-aminoadipic semialdehyde (AASA) and its Schiff base delta-1-piperideine-6-carboxylate (P6C). Although genetic testing of ALDH7A1 is the most definitive diagnostic method, quantifications of pathognomonic metabolites are important for the diagnosis and evaluation of therapeutic and dietary interventions. Current metabolite quantification methods use laborious, technically highly complex, and expensive liquid chromatography-tandem mass spectro-metry, which is available only in selected laboratories worldwide. Incubation of ortho-aminobenzaldehyde (oABA) with P6C leads to the formation of a triple aromatic ring structure with characteristic absorption and fluorescence properties. The mean concentration of P6C in nine urine samples from seven ATQ-deficient patients under standard treatment protocols was statistically highly significantly different (P < .001) compared to the mean of 74 healthy controls aged between 2 months and 57 years. Using this limited data set the specificity and sensitivity is 100% for all tested age groups using a P6C cut-off of 2.11 μmol/mmol creatinine, which represents the 99% prediction interval of the P6C concentrations in 17 control urine samples from children below 6 years of age. Plasma P6C concentrations were only elevated in one ATQ subject, possibly because P6C is trapped by pyridoxal-5-phosphate (PLP) blocking fusing with oABA. Nevertheless, both urine and plasma samples were amenable to the quantification of exogenous P6C with high response rates. The P6C quantification method using fusion of oABA with P6C is fast, simple, and inexpensive and might be readily implemented into routine clinical diagnostic laboratories for the early diagnosis of neonatal pyridoxine-dependent epilepsy.
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http://dx.doi.org/10.1002/jimd.12214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384183PMC
July 2020

Type I Interferon Signaling Disrupts the Hepatic Urea Cycle and Alters Systemic Metabolism to Suppress T Cell Function.

Immunity 2019 12 26;51(6):1074-1087.e9. Epub 2019 Nov 26.

CeMM Research Center for Molecular Medicine or the Austrian Academy of Sciences, Lazarettgasse 14 AKH BT25.3, 1090 Vienna, Austria. Electronic address:

Infections induce complex host responses linked to antiviral defense, inflammation, and tissue damage and repair. We hypothesized that the liver, as a central metabolic hub, may orchestrate systemic metabolic changes during infection. We infected mice with chronic lymphocytic choriomeningitis virus (LCMV), performed RNA sequencing and proteomics of liver tissue, and integrated these data with serum metabolomics at different infection phases. Widespread reprogramming of liver metabolism occurred early after infection, correlating with type I interferon (IFN-I) responses. Viral infection induced metabolic alterations of the liver that depended on the interferon alpha/beta receptor (IFNAR1). Hepatocyte-intrinsic IFNAR1 repressed the transcription of metabolic genes, including Otc and Ass1, which encode urea cycle enzymes. This led to decreased arginine and increased ornithine concentrations in the circulation, resulting in suppressed virus-specific CD8 T cell responses and ameliorated liver pathology. These findings establish IFN-I-induced modulation of hepatic metabolism and the urea cycle as an endogenous mechanism of immunoregulation. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.immuni.2019.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926485PMC
December 2019

MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation.

Nat Genet 2019 06 27;51(6):990-998. Epub 2019 May 27.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.
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http://dx.doi.org/10.1038/s41588-019-0413-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952269PMC
June 2019

Sphingolipid-dependent Dscam sorting regulates axon segregation.

Nat Commun 2019 02 18;10(1):813. Epub 2019 Feb 18.

Department for Neurobiology, University of Vienna, Althanstrasse 14, A-1090, Vienna, Austria.

Neurons are highly polarized cells with distinct protein compositions in axonal and dendritic compartments. Cellular mechanisms controlling polarized protein sorting have been described for mature nervous system but little is known about the segregation in newly differentiated neurons. In a forward genetic screen for regulators of Drosophila brain circuit development, we identified mutations in SPT, an evolutionary conserved enzyme in sphingolipid biosynthesis. Here we show that reduced levels of sphingolipids in SPT mutants cause axonal morphology defects similar to loss of cell recognition molecule Dscam. Loss- and gain-of-function studies show that neuronal sphingolipids are critical to prevent aggregation of axonal and dendritic Dscam isoforms, thereby ensuring precise Dscam localization to support axon branch segregation. Furthermore, SPT mutations causing neurodegenerative HSAN-I disorder in humans also result in formation of stable Dscam aggregates and axonal branch phenotypes in Drosophila neurons, indicating a causal link between developmental protein sorting defects and neuronal dysfunction.
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http://dx.doi.org/10.1038/s41467-019-08765-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379420PMC
February 2019

Ldlr and ApoE mice better mimic the human metabolite signature of increased carotid intima media thickness compared to other animal models of cardiovascular disease.

Atherosclerosis 2018 09 21;276:140-147. Epub 2018 Jul 21.

Institute of Cardiovascular and Metabolic Disease, Institut National de La Santé et de La Recherche Médicale (INSERM), Toulouse, France; Université Toulouse III Paul-Sabatier, Toulouse, France.

Background And Aims: Preclinical experiments on animal models are essential to understand the mechanisms of cardiovascular disease (CVD). Metabolomics allows access to the metabolic perturbations associated with CVD in heart and vessels. Here we assessed which potential animal CVD model most closely mimics the serum metabolite signature of increased carotid intima-media thickness (cIMT) in humans, a clinical parameter widely accepted as a surrogate of CVD.

Methods: A targeted mass spectrometry assay was used to quantify and compare a series of blood metabolites between 1362 individuals (KORA F4 cohort) and 5 animal CVD models: ApoE, Ldlr, and klotho-hypomorphic mice (kl/kl) and SHRSP rats with or without salt feeding. The metabolite signatures were obtained using linear regressions adjusted for various co-variates.

Results: In human, increased cIMT [quartile Q4 vs. Q1] was associated with 26 metabolites (9 acylcarnitines, 2 lysophosphatidylcholines, 9 phosphatidylcholines and 6 sphingomyelins). Acylcarnitines correlated preferentially with serum glucose and creatinine. Phospholipids correlated preferentially with cholesterol (total and LDL). The human signature correlated positively and significantly with Ldlr and ApoE mice, while correlation with kl/kl mice and SHRP rats was either negative and non-significant. Human and Ldlr mice shared 11 significant metabolites displaying the same direction of regulation: 5 phosphatidylcholines, 1 lysophosphatidylcholines, 5 sphingomyelins; ApoE mice shared 10.

Conclusions: The human cIMT signature was partially mimicked by Ldlr and ApoE mice. These animal models might help better understand the biochemical and molecular mechanisms involved in the vessel metabolic perturbations associated with, and contributing to metabolic disorders in CVD.
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http://dx.doi.org/10.1016/j.atherosclerosis.2018.07.024DOI Listing
September 2018

Altered membrane rigidity via enhanced endogenous cholesterol synthesis drives cancer cell resistance to destruxins.

Oncotarget 2018 May 22;9(39):25661-25680. Epub 2018 May 22.

Institute of Cancer Research, Department of Internal Medicine I, Medical University of Vienna, Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria.

Destruxins, secondary metabolites of entomopathogenic fungi, exert a wide variety of interesting characteristics ranging from antiviral to anticancer effects. Although their mode of action was evaluated previously, the molecular mechanisms of resistance development are unknown. Hence, we have established destruxin-resistant sublines of HCT116 colon carcinoma cells by selection with the most prevalent derivatives, destruxin (dtx)A, dtxB and dtxE. Various cell biological and molecular techniques were applied to elucidate the regulatory mechanisms underlying these acquired and highly stable destruxin resistance phenotypes. Interestingly, well-known chemoresistance-mediating ABC efflux transporters were not the major players. Instead, in dtxA- and dtxB-resistant cells a hyper-activated mevalonate pathway was uncovered resulting in increased cholesterol synthesis rates and elevated levels of lanosterol, cholesterol as well as several oxysterol metabolites. Accordingly, inhibition of the mevalonate pathway at two different steps, using either statins or zoledronic acid, significantly reduced acquired but also intrinsic destruxin resistance. Vice versa, cholesterol supplementation protected destruxin-sensitive cells against their cytotoxic activity. Additionally, an increased cell membrane adhesiveness of dtxA-resistant as compared to parental cells was detected by atomic force microscopy. This was paralleled by a dramatically reduced ionophoric capacity of dtxA in resistant cells when cultured in absence but not in presence of statins. Summarizing, our results suggest a reduced ionophoric activity of destruxins due to cholesterol-mediated plasma membrane re-organization as molecular mechanism underlying acquired destruxin resistance in human colon cancer cells. Whether this mechanism might be valid also in other cell types and organisms exposed to destruxins e.g. as bio-insecticides needs to be evaluated.
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http://dx.doi.org/10.18632/oncotarget.25432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986646PMC
May 2018

Targeted metabolomics and medication classification data from participants in the ADNI1 cohort.

Sci Data 2017 10 17;4:170140. Epub 2017 Oct 17.

Department of Psychiatry and Behavioral Sciences, and the Duke Institute for Brain Sciences, Duke University, Durham, NC 27710, USA.

Alzheimer's disease (AD) is the most common neurodegenerative disease presenting major health and economic challenges that continue to grow. Mechanisms of disease are poorly understood but significant data point to metabolic defects that might contribute to disease pathogenesis. The Alzheimer Disease Metabolomics Consortium (ADMC) in partnership with Alzheimer Disease Neuroimaging Initiative (ADNI) is creating a comprehensive biochemical database for AD. Using targeted and non- targeted metabolomics and lipidomics platforms we are mapping metabolic pathway and network failures across the trajectory of disease. In this report we present quantitative metabolomics data generated on serum from 199 control, 356 mild cognitive impairment and 175 AD subjects enrolled in ADNI1 using AbsoluteIDQ-p180 platform, along with the pipeline for data preprocessing and medication classification for confound correction. The dataset presented here is the first of eight metabolomics datasets being generated for broad biochemical investigation of the AD metabolome. We expect that these collective metabolomics datasets will provide valuable resources for researchers to identify novel molecular mechanisms contributing to AD pathogenesis and disease phenotypes.
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http://dx.doi.org/10.1038/sdata.2017.140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5644370PMC
October 2017

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma.

J Lipid Res 2017 12 6;58(12):2275-2288. Epub 2017 Oct 6.

Lipidomics Core Facility and Department of Pathology, Wayne State University, Detroit, MI.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.
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http://dx.doi.org/10.1194/jlr.M079012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711491PMC
December 2017

Metabolomic profiling of ascending thoracic aortic aneurysms and dissections - Implications for pathophysiology and biomarker discovery.

PLoS One 2017 3;12(5):e0176727. Epub 2017 May 3.

Cardiac Surgery Research Laboratory, University Clinic for Cardiac Surgery, Innsbruck Medical University, Innsbruck, Austria.

Objective: Our basic understanding of ascending thoracic aortic aneurysm (ATAA) pathogenesis is still very limited, hampering early diagnosis, risk prediction, and development of treatment options. "Omics"-technologies, ideal to reveal tissue alterations from the normal physiological state due to disease have hardly been applied in the field. Using a metabolomic approach, with this study the authors seek to define tissue differences between controls and various forms of ATAAs.

Methods: Using a targeted FIA-MS/MS metabolomics approach, we analysed and compared the metabolic profiles of ascending thoracic aortic wall tissue of age-matched controls (n = 8), bicuspid aortic valve-associated aneurysms (BAV-A; n = 9), tricuspid aortic valve-associated aneurysms (TAV-A; n = 14), and tricuspid aortic valve-associated aortic dissections (TAV-Diss; n = 6).

Results: With sphingomyelin (SM) (OH) C22:2, SM C18:1, SM C22:1, and SM C24:1 only 4 out of 92 detectable metabolites differed significantly between controls and BAV-A samples. Between controls and TAV-Diss samples only phosphatidylcholine (PC) ae C32:1 differed. Importantly, our analyses revealed a general increase in the amount of total sphingomyelin levels in BAV-A and TAV-Diss samples compared to controls.

Conclusions: Significantly increased levels of sphingomyelins in BAV-A and TAV-Diss samples compared to controls may argue for a repression of sphingomyelinase activity and the sphingomyelinase-ceramide pathway, which may result in an inhibition of tissue regeneration; a potential basis for disease initiation and progression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176727PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415060PMC
September 2017

Increased urine acylcarnitines in diabetic ApoE-/- mice: Hydroxytetradecadienoylcarnitine (C14:2-OH) reflects diabetic nephropathy in a context of hyperlipidemia.

Biochem Biophys Res Commun 2017 05 7;487(1):109-115. Epub 2017 Apr 7.

Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut of Cardiovascular and Metabolic Disease, France; Université Toulouse III Paul-Sabatier Toulouse, France. Electronic address:

Hyperlipidemia is a risk factor for initiation and progression of diabetic nephropathy but the metabolic pathways altered in the diabetic kidney in a context of hyperlipidemia remain incompletely described. Assuming that changes in urine composition reflect the alteration of renal metabolism and function, we analyzed the urine metabolite composition of diabetic (streptozotocin-treatment) and control (non diabetic) ApoE-/- mice fed a high cholesterol diet using targeted quantitative metabolomics. Urine metabolome was also compared to the plasma metabolome of the same animals. As previously shown, urine albuminuria/urine creatinine ratio (uACR) and glomerular area and plasma lipids (cholesterol, triglycerides) were more elevated in diabetic mice compared to control. After adjustment to urine creatinine, the abundance of 52 urine metabolites was significantly different in diabetic mice compared to control. Among them was a unique metabolite, C14:2-OH (3-hydroxytetradecadienoylcarnitine) that, in diabetic mice, was positively and significantly correlated with uACR, glomerular hypertrophy, blood glucose and plasma lipids. That metabolite was not detected in plasma. C14:2-OH is a long-chain acylcarnitine reminiscent of altered fatty acid beta oxidation. Other acylcarnitines, particularly the short chains C3-OH, C3-DC, C4:1, C5-DC, C5-M-DC, C5-OH that are reminiscent of altered oxidation of branched and aromatic amino acids were also exclusively detected in urine but were only correlated with plasma lipids. Finally, the renal gene expression of several enzymes involved in fatty acid and/or amino acid oxidation was significantly reduced in diabetic mice compared to control. This included the bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA (Ehhadh) that might play a central role in C14:2-OH production. This study indicate that the development of diabetes in a context of hyperlipidemia is associated with a reduced capacity of kidney to oxidize fatty acids and amino acids with the consequence of an elevation of urinary acetylcarnitines including C14:2-OH that specifically reflects diabetic nephropathy.
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http://dx.doi.org/10.1016/j.bbrc.2017.04.026DOI Listing
May 2017

Metabolic network failures in Alzheimer's disease: A biochemical road map.

Alzheimers Dement 2017 Sep 22;13(9):965-984. Epub 2017 Mar 22.

Department of Psychiatry, Duke University, Durham, NC, USA; Duke Institute for Brain Sciences, Duke University, Durham, NC, USA; Department of Medicine, Duke University Medical Center, Durham, NC, USA. Electronic address:

Introduction: The Alzheimer's Disease Research Summits of 2012 and 2015 incorporated experts from academia, industry, and nonprofit organizations to develop new research directions to transform our understanding of Alzheimer's disease (AD) and propel the development of critically needed therapies. In response to their recommendations, big data at multiple levels are being generated and integrated to study network failures in disease. We used metabolomics as a global biochemical approach to identify peripheral metabolic changes in AD patients and correlate them to cerebrospinal fluid pathology markers, imaging features, and cognitive performance.

Methods: Fasting serum samples from the Alzheimer's Disease Neuroimaging Initiative (199 control, 356 mild cognitive impairment, and 175 AD participants) were analyzed using the AbsoluteIDQ-p180 kit. Performance was validated in blinded replicates, and values were medication adjusted.

Results: Multivariable-adjusted analyses showed that sphingomyelins and ether-containing phosphatidylcholines were altered in preclinical biomarker-defined AD stages, whereas acylcarnitines and several amines, including the branched-chain amino acid valine and α-aminoadipic acid, changed in symptomatic stages. Several of the analytes showed consistent associations in the Rotterdam, Erasmus Rucphen Family, and Indiana Memory and Aging Studies. Partial correlation networks constructed for Aβ, tau, imaging, and cognitive changes provided initial biochemical insights for disease-related processes. Coexpression networks interconnected key metabolic effectors of disease.

Discussion: Metabolomics identified key disease-related metabolic changes and disease-progression-related changes. Defining metabolic changes during AD disease trajectory and its relationship to clinical phenotypes provides a powerful roadmap for drug and biomarker discovery.
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http://dx.doi.org/10.1016/j.jalz.2017.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866045PMC
September 2017

Blood metabolite markers of preclinical Alzheimer's disease in two longitudinally followed cohorts of older individuals.

Alzheimers Dement 2016 07 21;12(7):815-22. Epub 2016 Jan 21.

Clinical and Translational Neuroscience Unit, Laboratory of Behavioral Neuroscience, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA. Electronic address:

Introduction: Recently, quantitative metabolomics identified a panel of 10 plasma lipids that were highly predictive of conversion to Alzheimer's disease (AD) in cognitively normal older individuals (n = 28, area under the curve [AUC] = 0.92, sensitivity/specificity of 90%/90%).

Methods: Quantitative targeted metabolomics in serum using an identical method as in the index study.

Results: We failed to replicate these findings in a substantially larger study from two independent cohorts-the Baltimore Longitudinal Study of Aging ([BLSA], n = 93, AUC = 0.642, sensitivity/specificity of 51.6%/65.7%) and the Age, Gene/Environment Susceptibility-Reykjavik Study ([AGES-RS], n = 100, AUC = 0.395, sensitivity/specificity of 47.0%/36.0%). In analyses applying machine learning methods to all 187 metabolite concentrations assayed, we find a modest signal in the BLSA with distinct metabolites associated with the preclinical and symptomatic stages of AD, whereas the same methods gave poor classification accuracies in the AGES-RS samples.

Discussion: We believe that ours is the largest blood biomarker study of preclinical AD to date. These findings underscore the importance of large-scale independent validation of index findings from biomarker studies with relatively small sample sizes.
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http://dx.doi.org/10.1016/j.jalz.2015.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947451PMC
July 2016

The ratio of phosphatidylcholines to lysophosphatidylcholines in plasma differentiates healthy controls from patients with Alzheimer's disease and mild cognitive impairment.

Alzheimers Dement (Amst) 2015 Sep;1(3):295-302

Department of Psychiatry and Psychotherapy, University Clinic of General and Social Psychiatry, Medical University of Innsbruck, Innsbruck, Austria.

Background: Metabolomic processes have been identified as being strongly linked to the development of Alzheimer's disease (AD). Thus, lipid metabolites appear to be highly useful as diagnostic substrates for the diagnosis of AD and mild cognitive impairment (MCI) in plasma.

Methods: We analyzed plasma samples from controls (n = 35), MCI (n = 33), and AD patients (n = 43) using the AbsoluteIDQ p180 Kit (Biocrates Life Sciences), which included quantitative analysis of 40 acylcarnitines, 21 amino acids, 19 biogenic amines, 15 sphingolipids, 90 glycerophospholipids, and sum of hexoses.

Results: We found that individual lipid metabolites can differentiate controls from MCI and AD with relevant significance. However, the ratio between PC aa C34:4 and lysoPC a C18:2 differentiates controls from MCI ( = .0000007) and from AD ( = .0000009) with greater significance.

Conclusions: The results provide evidence that the ratio of these two lipid metabolites is useful for diagnosing MCI and AD with an accuracy of 82%-85%.
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http://dx.doi.org/10.1016/j.dadm.2015.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700585PMC
September 2015

Systems-level organization of yeast methylotrophic lifestyle.

BMC Biol 2015 Sep 23;13:80. Epub 2015 Sep 23.

Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190, Vienna, Austria.

Background: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date.

Results: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation.

Conclusions: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology.
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http://dx.doi.org/10.1186/s12915-015-0186-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580311PMC
September 2015

Sphingomyelin SM(d18:1/18:0) is significantly enhanced in cerebrospinal fluid samples dichotomized by pathological amyloid-β42, tau, and phospho-tau-181 levels.

J Alzheimers Dis 2015 ;44(4):1193-201

Department of Psychiatry and Psychotherapy, University Clinic of General and Social Psychiatry, Medical University of Innsbruck, Innsbruck, Austria.

Alzheimer's disease (AD) is a severe and chronic neurodegenerative disorder of the brain. The laboratory diagnosis is limited to the analysis of three biomarkers in cerebrospinal fluid (CSF): amyloid-β42 (Aβ42), total tau, and phospho-tau-181 (P-tau-181). However, there is a need to find more biomarkers in CSF that can improve the sensitivity and specificity. The aim of the present study was to analyze endogenous small metabolites (metabolome) in the CSF, which may provide potentially new insights into biochemical processes involved in AD. One hundred CSF samples were dichotomized by normal (n = 50) and pathological decreased Aβ42 and increased tau and P-tau-181 levels (n = 50; correlating to an AD-like pathology). These CSF samples were analyzed using the AbsoluteIDQ® p180 Kit (BIOCRATES Life Sciences), which included 40 acylcarnitines, 21 amino acids, 19 biogenic amines, 15 sphingolipids, and 90 glycerophospholipids. Our data show that two sphingomyelins (SM (d18:1/18:0) and SM (d18:1/18:1)), 5 glycerophospholipids (PC aa C32:0, PC aa C34:1, PC aa C36:1, PC aa C38:4 and PC aa C38:6), and 1 acylcarnitine (C3-DC-M/C5-OH) were significantly altered in the CSF with pathological "AD-like pathology". Sphingomyelin SM (d18:1/18:0) proved to be a specific (76%) and sensitive (66%) biomarker with a defined cut-off of 546 nM. Correct diagnoses for 21 out of 32 unknown samples could be achieved using this SM (d18:1/18:0) cut-off value. In conclusion, the sphingolipid SM (d18:1/18:0) is significantly increased in CSF of patients displaying pathological levels of Aβ42, tau, and P-tau-181.
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http://dx.doi.org/10.3233/JAD-142319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699259PMC
November 2015

Quantitative metabolite profiling utilizing parallel column analysis for simultaneous reversed-phase and hydrophilic interaction liquid chromatography separations combined with tandem mass spectrometry.

Anal Chem 2014 May 8;86(9):4145-50. Epub 2014 Apr 8.

Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences, BOKU-Vienna , Muthgasse 18, 1190 Vienna, Austria.

In this work, a fully automated parallel LC column method was established in order to perform orthogonal hydrophilic interaction chromatography (HILIC) and reversed-phase (RPLC) chromatography within one analytical run for targeted quantitative mass spectrometric determination of metabolites from central carbon metabolism. In this way, the analytical throughput could be significantly improved compared to previously established dual separation work flows involving two separate analytical runs. Two sample aliquots were simultaneously injected onto a dual column setup columns using a ten-port valve, and parallel separations were carried out. Sub 2 μm particle size stationary phases were employed for both separation methods. HILIC and RPLC eluents were combined post column followed by ESI-MS/MS detection. The orthogonal separations were optimized, aiming at an overall separation with 2 retention time segments, while reversed-phase separation was accomplished within 5.5 min; metabolites on the HILIC phase were retained for a minimum time of 6 min. The overall run time was 15 min. The setup was applied to the quantification of 30 primary intercellular metabolites, including amino acids, organic acids, and nucleotides employing internal standardization by a fully (13)C-labeled yeast extract. The comparison with HILIC-MS/MS and RPLC-MS/MS in separate analytical runs revealed that an excellent analytical performance was achieved by the parallel LC column method. The experimental repeatability (N = 5) was on average <5% (only for 2 compounds >5%). Moreover, limits of detection for the new approach ranging from 0.002-15 μM were in a good agreement with ones obtained in separate HILIC-MS/MS and RPLC-MS/MS runs (ranging from 0.01-44 μM).
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http://dx.doi.org/10.1021/ac5003454DOI Listing
May 2014

Fully automated on-line two-dimensional liquid chromatography in combination with ESI MS/MS detection for quantification of sugar phosphates in yeast cell extracts.

Analyst 2014 Mar;139(6):1512-20

University of Natural Resources and Life Sciences, BOKU-Vienna, Department of Chemistry, Division of Analytical Chemistry, Muthgasse 18, 1190, Vienna, Austria.

A mass spectrometric quantitative assay was developed for the analysis of 10 sugar phosphates in the yeast Pichia pastoris. As a novelty, two-dimensional chromatography based on a fully automated heart-cutting LC-LC technique was introduced. Using a ten-port valve, ten fractions of the first chromatographic dimension, i.e. anion exchange chromatography (AEC), were transferred and separated by the orthogonal second dimension, i.e. separation on porous graphitized carbon. The chromatographic separation on the second dimension was optimized for each transferred fraction minimizing the separation time and ensuring complete removal of the salt constituents of the AEC eluents. The latter being crucial for electrospray mass spectrometric detection was confirmed by combining the LC-LC separation with on-line ICP-MS detection. These measurements showed that sodium elution was completed after 0.8 min. Consequently, an analysis time of 1 min per transferred peak was established. In this way, the excellent peak capacity given by ion exchange could be conserved in the second dimension at the same time enabling mass spectrometric detection. Sub-μM limits of detection could be obtained by the new LC-LC-MS/MS methods ranging between 0.03 and 0.19 μM for the investigated compounds (only 3GAP showed a LOD of 1 μM). The method was applied to the quantification of ten sugar phosphates in yeast extracts utilizing internal standardization with a fully labeled (13)C yeast extract. Typically, the standard uncertainties for N = 3 replicates assessed by the LC-LC-MS/MS set-up were <5%.
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http://dx.doi.org/10.1039/c3an01930fDOI Listing
March 2014

Interlaboratory comparison for quantitative primary metabolite profiling in Pichia pastoris.

Anal Bioanal Chem 2013 Jun 19;405(15):5159-69. Epub 2013 Apr 19.

Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria.

For the first time, an interlaboratory comparison was performed in the field of quantitative metabolite profiling in Pichia pastoris. The study was designed for the evaluation of different measurement platforms integrating different quantification strategies using internal standardization. Nineteen primary metabolites including amino acids and organic acids were selected for the study. Homogenous samples were obtained from chemostat fermentations after rapid sampling, quenching and filtration, and hot ethanol extraction. Laboratory 1 (BOKU) employed an in vivo-synthesized fully labeled U(13)C cell extracts of P. pastoris for immediate internal standardization upon cell extraction. Quantification was carried out using orthogonal reversed-phase (RP-LC) and hydrophilic interaction chromatography (HILIC) in combination with tandem mass spectrometry. Laboratory 2 (Biocrates) applied a metabolomics kit allowing fully automated, rapid derivatization, solid phase extraction and internal standardization in 96-well plates with immobilized isotopically enriched internal standards in combination with HILIC-MS-MS and RP-LC-MS-MS for organic acids and derivatized amino acids, respectively. In this study, the obtained intracellular concentrations ranged from 0.2 to 108 μmol g(-1) cell dry weight. The total combined uncertainty was estimated including uncertainty contributions from the corresponding MS-based measurement and sample preparation for each metabolite. Evidently, the uncertainty contribution of sample preparation was lower for the values obtained by laboratory 1, implementing isotope dilution upon extraction. Total combined uncertainties (K = 2) ranging from 21 to 48% and from 30 to 57% were assessed for the quantitative results obtained in laboratories 1 and 2, respectively. The major contribution arose from sample preparation, hence from repeatability precision of the extraction procedure. Finally, the laboratory intercomparison was successful as most of the investigated metabolites showed concentration levels agreeing within their total combined uncertainty, implying that accurate quantification was given. The application of isotope dilution upon extraction was an absolute prerequisite for the quantification of the redox-sensitive amino acid methionine, where no agreement between the two laboratories could be achieved.
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http://dx.doi.org/10.1007/s00216-013-6964-4DOI Listing
June 2013

U13C cell extract of Pichia pastoris--a powerful tool for evaluation of sample preparation in metabolomics.

J Sep Sci 2012 Nov 22;35(22):3091-105. Epub 2012 Oct 22.

Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria.

Quantitative metabolic profiling is preceded by dedicated sample preparation protocols. These multistep procedures require detailed optimization and thorough validation. In this work, a uniformly (13)C-labeled (U(13)C) cell extract was used as a tool to evaluate the recoveries and repeatability precisions of the cell extraction and the extract treatment. A homogenous set of biological replicates (n = 15 samples of Pichia pastoris) was prepared for these fundamental experiments. A range of less than 30 intracellular metabolites, comprising amino acids, nucleotides, and organic acids were measured both in monoisotopic (12)C and U(13)C form by LC-MS/MS employing triple quadrupole MS, reversed phase chromatography, and HILIC. Recoveries of the sample preparation procedure ranging from 60 to 100% and repeatability precisions below 10% were obtained for most of the investigated metabolites using internal standardization approaches. Uncertainty budget calculations revealed that for this complex quantification task, in the optimum case, total combined uncertainty of 12% could be achieved. The optimum case would be represented by metabolites, easy to extract from yeast with high and precise recovery. In other cases the total combined uncertainty was significantly higher.
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http://dx.doi.org/10.1002/jssc.201200447DOI Listing
November 2012

Collision-induced dissociation of imidazolium-based zwitterionic liquids.

Eur J Mass Spectrom (Chichester) 2009 ;15(4):471-8

Faculty of Chemistry, University of Latvia, Riga, Latvia.

Fragmentation pathways of some imidazolium based zwitterionic liquids-3-(3-alkyl-1-imidazolio)- propane sulfonates and 3-(2-methyl-3-alkyl-1-imidazolio)-propane sulfonates -- have been studied by tandem electrospray mass spectrometry and collision-induced dissociation. The relative abundances of the lowest energy fragment ions depend on the length of the alkyl chain at the (II)N of the imidazolium ring and the cone voltage. The first fragment ions originate from the scission of C(non aromatic)-N bond of compounds investigated, but with increasing collision energy, scission of C-C bonds occurs. Aggregates of the general formula [(M + H)(x) + (M)(y)](+) (x;y = 1-2) formed. Methyl substituted zwitterionic liquids show higher molecular stability than 3-(3-alkyl-1-imidazolio)-propane sulfonates.
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http://dx.doi.org/10.1255/ejms.997DOI Listing
September 2009