Metabolomics of hay fever and the Metabolomics of hay fever and the pathophysiology of sneezingpathophysiology of sneezing

Klaus M. Weinberger, Ph.D.Klaus M. Weinberger, Ph.D.

www.biocrates.at

EN ISO 9001:2000EN ISO 9001:2000

life sciences

Early spring in AustriaEarly spring in Austria

Simple causal relationships...Simple causal relationships...

Disabled speakerDisabled speaker

Targeted metabolomics in Targeted metabolomics in pharmacodynamics and toxicologypharmacodynamics and toxicology

Klaus M. Weinberger, Ph.D.Klaus M. Weinberger, Ph.D.

www.biocrates.at

EN ISO 9001:2000EN ISO 9001:2000

life sciences

DNADNA RNARNA PolypeptidesPolypeptides ProteinsProteins

MetabolitesMetabolites

Tran

scrip

tion

Tran

scrip

tion

Tran

slatio

nTr

ansla

tion

PTM

PTM

EnzymaticEnzymatic activityactivity

Metabolomics in Metabolomics in systemssystems biologybiology

•• FunctionalFunctional endend--pointpoint of of geneticgeneticinformationinformation

•• Mirroring externalMirroring external influencesinfluences byby::•• ((MalMal--)nutrition)nutrition•• PhysicalPhysical activityactivity•• MedicationMedication

life sciences

CitCit

ArgArgOrnOrn

ArgsuccArgsucc

FumFum

UreaUrea

AspAsp

CarbCarb--PP

NONO

NOSNOS

ASLASL

ASSASS

ARGARG

OCTOCT

Linking metabolites to enzymes and pathwaysLinking metabolites to enzymes and pathways

life sciences

Discovery

Validation

Application

Biomarker technologies

NMRNMRGCxGC/MSGCxGC/MS

LCLC--MS (full ion scan)MS (full ion scan)

LCLC--MS/MSMS/MSFIAFIA--MS/MSMS/MS

Precursor ion and Precursor ion and neutral loss scansneutral loss scans

ProfilingIdentification

TargetedQuantitation

LCLC--MS/MSMS/MSFIAFIA--MS/MSMS/MS

Multiple reaction monitoringMultiple reaction monitoring

Number of parameters

Number ofsamples

life sciences

Integrated metabolomics platformIntegrated metabolomics platform

•• Automated separation and Automated separation and derivatizationderivatization

•• Solid phase extractionSolid phase extraction•• Alliance with Hamilton Alliance with Hamilton

RoboticsRobotics

Sample preparationSample preparation•• Technical validationTechnical validation•• Statistical analysisStatistical analysis•• Data visualizationData visualization•• Biochemical interpretationBiochemical interpretation•• Partnership with Partnership with GeneGoGeneGo

BioInformaticsBioInformatics

diseas

e 1

disease2

Clinical & experimentalClinical & experimentalsamplessamples

Diagnoses & lab dataDiagnoses & lab data

BioBankBioBank

LIMS/DatabaseLIMS/Database

•• IdentificationIdentification•• QuantitationQuantitation•• QA/QCQA/QC•• Alliance with Applied Alliance with Applied

BiosystemsBiosystems / MDS / MDS SciexSciex

AnalyticsAnalytics

life sciences

Analytical portfolioAnalytical portfolio• Primary and secondary amines

– proteinogenic and non-proteinogenic amino acids– alkylated and nitrated amino acids– biogenic amines and polyamines

• Acylcarnitines and free carnitine• Reducing mono- and oligosaccharides• Phospho- and glycolipids

– PC, PE, PS, PG, PA, PI(P), Cer, SM, ...• Eicosanoids & other oxidized fatty acids (COX, LOX activity; non-enzymatic

oxidation)– Prostaglandins, prostacyclins, thromboxanes– Leukotrienes, HODEs, HETEs, ...

• Bile acids & their conjugates• Energy metabolism intermediates

– Phosphorylated sugars– Nucleotides– Mono-, di-, trivalent organic acids

Routine quantitation of > 1000 annotated metabolites

covering main areas of intermediary metabolism

life sciences

Areas of applicationAreas of applicationØØ Basic researchBasic research

-- Beyond genomics, transcriptomics and proteomics in cell biology,Beyond genomics, transcriptomics and proteomics in cell biology,oncology, inflammation, etc.oncology, inflammation, etc.

ØØ Agricultural & nutrition industryAgricultural & nutrition industry-- Plant intermediary metabolismPlant intermediary metabolism-- Health effects of functional foodHealth effects of functional food-- NutrigenomicsNutrigenomics

ØØ Clinical diagnostics & theranosticsClinical diagnostics & theranostics-- ProofProof--ofof--concept in neonatal screeningconcept in neonatal screening-- Early diagnosis and accurate staging of complex diseasesEarly diagnosis and accurate staging of complex diseases-- Specific monitoring of therapeutic effectsSpecific monitoring of therapeutic effects

ØØ Pharmaceutical R&DPharmaceutical R&D-- Drug metabDrug metabolism / Pharmacokineticsolism / Pharmacokinetics-- Safety / ToxSafety / Tox-- Pharmacodynamics / EfficacyPharmacodynamics / Efficacy

life sciences

MetabolitesMetabolites givegive insightinsight at different at different levelslevels

LipidLipid signallingsignalling ((PIsPIs, , PIPsPIPs, , Ceramides, SCeramides, S--11--PP etc.)etc.)Regulatory metabolites (ADMA/SDMA, Regulatory metabolites (ADMA/SDMA, FF--2,62,6--BP)BP)

IV.IV. ModeMode of of actionaction markersmarkers

Gluconeogenesis / GlycolysisGluconeogenesis / GlycolysisGlycogen synthesis / GlycogenolysisGlycogen synthesis / GlycogenolysisTissueTissue damage and apoptosisdamage and apoptosisOxidative stressOxidative stress

III.III. MultiparametricMultiparametric surrogatesurrogatemarkersmarkers

LipidLipid elevationelevation//loweringlowering; ; subsets!subsets!Metabolic control / Insulin sensitivityMetabolic control / Insulin sensitivityInflammation mediatorsInflammation mediatorsTubular dysfunctionTubular dysfunction

II.II. DirectDirect multiparametricmultiparametric markersmarkers

CarnitineCarnitine palmitoylpalmitoyl transferasetransferase IIStearoylStearoyl--CoACoA desaturasedesaturaseNO NO synthasesynthase

I.I. MarkersMarkers forfor activitiesactivities ofof singlesingleenzymesenzymes

life sciences

Use case: puromycinUse case: puromycin--induced nephrotoxicityinduced nephrotoxicity

Study design: 4 cohorts of 6 SpragueStudy design: 4 cohorts of 6 Sprague--Dawley ratsDawley rats

I.I. ControlsControls only vehicleonly vehicle

II.II. 10 mg/kg10 mg/kg no histopathologyno histopathology

III.III. 20 mg/kg20 mg/kg moderate nephrosismoderate nephrosis

IV.IV. 40 mg/kg40 mg/kg ESRD after two weeksESRD after two weeks

Plasma and urine at 4 timepoints: Plasma and urine at 4 timepoints:

Days 3, 7, 14, and 22Days 3, 7, 14, and 22

Correlated with histopathology, pathophysiology, expression Correlated with histopathology, pathophysiology, expression profiling, and proteomicsprofiling, and proteomics

life sciences

PalmitoylcarnitinePalmitoylcarnitine

General tissue damageGeneral tissue damage

ØØ DoseDose-- and timeand time--dependent increase in plasma acylcarnitine concentrationsdependent increase in plasma acylcarnitine concentrations

ØØ No preference for certain chain length, no depletion of free carNo preference for certain chain length, no depletion of free carnitinenitine

ØØ Established biomarker of tissue leakage and Established biomarker of tissue leakage and –– particularly particularly –– mitochondrial damagemitochondrial damage

life sciences

CDCACDCA

LCALCA

CACA

DCADCA

UDCAUDCA

No overt hepatotoxicityNo overt hepatotoxicity

life sciences

Arachidonic acidArachidonic acid12(S)12(S)--HETEHETE 15(S)15(S)--HETEHETE

1212--LipoxygenaseLipoxygenase 1515--LipoxygenaseLipoxygenase

Oxidation of polyunsaturated fatty acidsOxidation of polyunsaturated fatty acids

life sciences

ØØ Increased ratios of prostaglandins Increased ratios of prostaglandins & thromboxanes to arachidonic & thromboxanes to arachidonic acidacid

ØØ PGE2 proPGE2 pro--diureticdiuretic

ØØ PGE2 and TXB2 associated with PGE2 and TXB2 associated with proteinuriaproteinuria

PGD2/AAPGD2/AA PGE2/AAPGE2/AA

TXB2/AATXB2/AA

DoseDose--dependant cyclooxygenase activationdependant cyclooxygenase activation

life sciences

SDMA levels in puromycinSDMA levels in puromycin--induced nephrosisinduced nephrosis

PlasmaPlasma UrineUrine

ØØ Established plasma marker of kidney Established plasma marker of kidney damagedamage

ØØ Higher predictive value than ADMAHigher predictive value than ADMAØØ Reportedly lower SDMA clearance in Reportedly lower SDMA clearance in

rat remnant kidney model not observed rat remnant kidney model not observed herehere

ØØ Increased activity of protein arginine Increased activity of protein arginine NN--methyltransferase IImethyltransferase II

life sciences

Time course in lowTime course in low--dose cohortdose cohort

life sciences

TimeTime--dependent lysinuriadependent lysinuria

ØØ No histopathological findings in 10 mg/kg groupNo histopathological findings in 10 mg/kg group

ØØ Separate resorption system for dibasic amino acids (Arg, Orn, LySeparate resorption system for dibasic amino acids (Arg, Orn, Lys)s)

ØØ Effects on Arg and Orn less pronounced (Cave: role in urea cycleEffects on Arg and Orn less pronounced (Cave: role in urea cycle))

life sciences

ØØ Direct toxicity / tissue damageDirect toxicity / tissue damageØØ Acylcarnitine leakage and release of membrane phospholipidsAcylcarnitine leakage and release of membrane phospholipidsØØ Activation of phospholipasesActivation of phospholipasesØØ No overt hepatotoxicityNo overt hepatotoxicity

ØØ Moderate polyuria and specific tubular dysfunctionModerate polyuria and specific tubular dysfunctionØØ Inflammation / oxidative stressInflammation / oxidative stress

ØØ DoseDose--dependant activation of COX, 12dependant activation of COX, 12--LOX and 15LOX and 15--LOXLOXØØ Increase of proIncrease of pro--inflammatory, proinflammatory, pro--diuretic eicosanoidsdiuretic eicosanoidsØØ No indication of systemic oxidative stressNo indication of systemic oxidative stress

ØØ Other markersOther markersØØ TimeTime--dependent moderate ketosisdependent moderate ketosisØØ DeDe--repression of NO synthaserepression of NO synthaseØØ Elevated plasma SDMA, in this model also elevated urine levelsElevated plasma SDMA, in this model also elevated urine levelsØØ Depletion of plasma tryptophan, but mainly conversion to Depletion of plasma tryptophan, but mainly conversion to

serotonin (instead of kynurenine); mediator for vasoconstrictionserotonin (instead of kynurenine); mediator for vasoconstrictionin kidneyin kidney

Study summaryStudy summary

life sciences

SemiSemi--standardizedstandardized

StandardizedStandardized

StandardizedStandardized

Sampling Sampling conditionsconditions

DiverseDiverseDiverseDiverseHomoHomo

ControlledControlledSimilarSimilar(inbred strains)(inbred strains)

CanisCanis

ControlledControlledIdenticalIdentical(clones)(clones)

MusMus

Biological Biological variabilityvariabilityNutritionNutritionGeneticsGeneticsGenusGenus

Concerns for clinical studiesConcerns for clinical studies

Observed effects significantly greater than Observed effects significantly greater than ‚‚normalnormal‘‘ biological variability?biological variability?

life sciences

CitCit

ArgArgOrnOrn

ArgsuccArgsucc

FumFum

UreaUrea

AspAsp

CarbCarb--PP

ASLASL

ASSASS

ARGARG

OCTOCT

Urea cycle in mild metabolic syndromeUrea cycle in mild metabolic syndrome

life sciences

healthy MetSyn MetSyn +CVD MetSyn - CVD

Arg

inin

e [µ

M]

2

4

6

8

10

12

14

16

p < 0.0001

Urea cycle alterations in metabolic syndrome

healthy MetSyn MetSyn +CVD MetSyn - CVD

Orn

ithin

e [µ

M]

0

2

4

6

8

10

12

14

p < 0.0001

ØØ highly significant and specific differences even in highly significant and specific differences even in small clinical study (n = 100)small clinical study (n = 100)

ØØ ratios of metabolites and pathway mapping yield ratios of metabolites and pathway mapping yield even clearer separationeven clearer separation

life sciences

Molecular pathophysiology

Molecular pharmacodynamics

Pharmacology Toxicology

PathologyPhysiology

Clinical chemistryMolecular Dx

Evidence-based drug development

Vision for drug developmentVision for drug development

EN ISO 9001:2000EN ISO 9001:2000

Pharmacology Toxicology

life sciences

AcknowledgementsAcknowledgements

EN ISO 9001:2000EN ISO 9001:2000

BioinformaticsBioinformaticsArmin Armin GraberGraberOlivier Olivier LefLefèèvrevrePaolo Paolo ZaccariaZaccariaFlorianFlorian BichtelerBichtelerMarc BreitMarc BreitKarl Karl KuglerKuglerBernd HaasBernd HaasMattias BairMattias BairRobert EllerRobert EllerHamza OvacinHamza OvacinGerd LorGerd LorüünsernserDaniel AndresDaniel Andres

AnalyticsAnalyticsStevenSteven RamsayRamsaySascha DammeierSascha DammeierKatussevaniKatussevani BernardoBernardoTherese KoalTherese KoalCornelia RCornelia RööhringhringWolfgang Wolfgang GuggenbichlerGuggenbichlerWolfgang Wolfgang StStöögglgglCarmen BurgmeierCarmen BurgmeierInes UnterwurzacherInes UnterwurzacherPeter Peter EnohEnohLisa KLisa KöörnerrnerDoreen KirchbergDoreen KirchbergVerena Verena ForcherForcher

StatisticsStatistics & & BiochemistryBiochemistryIngridIngrid OsprianOsprianMarion BeierMarion BeierGGüünther Eiblnther EiblGerhard MariniGerhard MariniMatthias KellerMatthias KellerOliverOliver LutzLutzSusanna OlscherSusanna Olscher

Admin & BDAdmin & BDBrad MorieBrad MorieAnton GronesAnton GronesDoris GigeleDoris GigeleIngrid SandnerIngrid SandnerThomas SiebererThomas Sieberer

Applied Biosystems / MDS SciexApplied Biosystems / MDS SciexJulie WingateJulie Wingate Karin WihsbKarin WihsbööckckBruno CasettaBruno Casetta Holm SommerHolm SommerJoe AnacletoJoe Anacleto Gisbert SchGisbert SchääferferByron Kieser Byron Kieser Bori ShushanBori ShushanDale Patterson Dale Patterson Lyle BurtonLyle BurtonMasahide HabuMasahide Habu Chris EliconeChris EliconeChristoph Kuntzsch Christoph Kuntzsch Ron BonnerRon Bonner

Subhasish PurkayasthaSubhasish Purkayastha

life sciences

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