犬尿酸作为精神分裂症标志物.pdf

Copyright 2014 American Medical Association All rights reserved Stress InducedIncreaseinKynurenicAcid asaPotentialBiomarkerforPatients WithSchizophreniaandDistressIntolerance JoshuaChiappelli MD AnaPocivavsek PhD KatieL Nugent PhD FrancescaM Notarangelo PhD PeterKochunov PhD LauraM Rowland PhD RobertSchwarcz PhD L ElliotHong MD IMPORTANCESeverallinesofevidencehavelinkedtheendogenousneuromodulator kynurenicacid KYNA toschizophrenia Thepathophysiologyofschizophreniaiscommonly associatedwithstress andstressplaysakeyregulatoryroleinthefirst rate limitingstepof thekynureninepathway whichproducesKYNA OBJECTIVETodeterminewhetherthelevelofKYNAchangesfollowingpsychologicalstress andwhetherthischangeisassociatedwithstress relatedbehavior DESIGN SETTING AND PARTICIPANTSTheKYNAlevelwasmeasuredinsalivasamplestakenat baselineandat2timesfollowingalaboratory basedpsychologicalstresschallengein128 participants 64patientswithschizophreniafromoutpatientclinicsand64healthycontrols fromthecommunity EXPOSURELaboratory basedpsychologicalstresschallenge MAIN OUTCOMES AND MEASURESQuittingthestressfultaskearlywasusedasabehavioral markerofdistressintolerance RESULTSPatientswithschizophreniashowedasignificantlyhigherrateofdistress intolerancecomparedwithhealthycontrols P 003 SalivaryKYNAlevelsincreased significantlybetweenbaselineand20minutesfollowingthestresstaskinbothpatientsand controls mean SEM 6 72nM 0 65nM vs8 43nM 1 05nM respectively P 007 Patientswhowereunabletotoleratethestressfultasksandquitearlyshowedsignificantly higherlevelsofKYNAthanpatientswhotoleratedthepsychologicalstressor P 02 or healthycontrols P 02 Inpatientswithdistressintolerance KYNAelevationsignificantly correlatedwiththeseverityofclinicalsymptoms 0 64 P 008 CONCLUSIONS AND RELEVANCEDistressintoleranceismorecommoninpatientswith schizophrenia PatientswiththisbehavioralphenotypehaveelevatedsalivaryKYNAlevels Thisstressresponsebehavior linkedbiomarkermayaidheterogeneityreductionin schizophreniaandotherstress relatedpsychiatricconditions JAMAPsychiatry doi 10 1001 jamapsychiatry 2014 243 PublishedonlineMay7 2014 Editorial Supplementalcontentat AuthorAffiliations Maryland PsychiatricResearchCenter DepartmentofPsychiatry University ofMarylandSchoolofMedicine Baltimore CorrespondingAuthor Joshua Chiappelli MD MarylandPsychiatric ResearchCenter POBox21247 Baltimore MD21228 jchiappelli mprc umaryland edu Research OriginalInvestigation E1 Copyright 2014 American Medical Association All rights reserved Downloaded From by a Yale University User on 05 16 2014 Copyright 2014 American Medical Association All rights reserved C onverginglinesofevidenceimplicateabnormalitiesin the kynurenine pathway KP of tryptophan degrada tioninschizophrenia 1 4Increasedconcentrationsofthe KP metabolites kynurenine and kynurenic acid KYNA have been found in postmortem brain tissue1 2and cerebrospinal fluid3 4of individuals with schizophrenia Moreover genetic variants in KP enzymes have been linked to psychotic symp toms cognitivedysfunctions andabnormallevelsofKYNA 5 7 In the brain KYNA can act as an antagonist at N methyl D aspartateand 7nicotinicreceptors bothofwhichhavebeen closely associated with schizophrenia 8At higher concentra tions KYNA can also be neuroprotective in conditions of excitotoxicity 9 11However the clinical significance of kynu renergic abnormalities in patients with schizophrenia re mains unclear Therate limitingstepoftheKPistheinitialconversionof tryptophantokynurenine whichiscontrolledby2enzymes tryptophan 2 3 dioxygenase TDO and indoleamine 2 3 dioxygenase IDO 1 and IDO 2 12 13Then KYNA is produced fromkynureninethroughirreversibletransaminationbykyn urenine aminotransferases see the article by Schwarcz et al8 for review Stress broadly defined as environmental factors that threaten well being and demand a homeostatic response from an individual 14has been implicated in all stages of schizophrenia Specifically stress is a risk factor in the neu rodevelopmental etiology of schizophrenia during preg nancy 15Stress is also associated with the onset and progres sion of symptoms in adolescence16and is often linked to exacerbation of psychotic symptoms in adulthood 17In ani mal models stressors robustly induce TDO activity18 19and thereby stimulate the formation of KYNA and other KP metabolites 20 21in part through activation of the hypo thalamic pituitary adrenal axis 22 23Stress also increases kyn urenines through IDO an enzyme typically regulated by cytokines For example acoustic stress and restraint stress in animals induce IDO gene expression and the subsequent increase in kynurenine and KYNA levels and this effect is blocked by pretreatment with antibodies to inflammatory cytokines 24In humans the stress of anticipating surgery was found to be associated with an increased plasma kyn urenine to tryptophan ratio suggestive of increased IDO activity 24These findings collectively suggest the existence of pathways by which stress can increase the formation of KYNA and other KP metabolites Thus the KP with TDO and IDO as critical points of regulation is a potential nexus for mechanisms linking stress to clinical dysfunctions in schizophrenia However direct evidence of stress responsiveness of this pathway in schizophrenia is lacking In this study we aimed to investi gate responsiveness of KYNA to psychological stress in people with and without schizophrenia and to test the hypothesis that a KYNA response to stress can serve as a bio marker for maladaptive stress related behaviors in the dis ease Using a noninvasive method to functionally interrogate this pathway we examined KYNA reactivity in saliva col lected before and after a laboratory based psychological stress task Methods Participants PatientswererecruitedfromtheoutpatientclinicsattheMary land Psychiatric Research Center and neighboring mental health clinics Healthy controls were recruited through me diaadvertisementsandrandomdigitdialing Diagnoseswere confirmed with the Structured Clinical Interview for DSM IV in all participants Major medical and neurological illnesses history of head injury with cognitive sequelae mental retar dation substancedependencewithinthepast6months orcur rentsubstanceabuse exceptnicotine wereexclusionary Con trols had no current DSM IV Axis I diagnoses and no family historyofpsychosisintheprior2generations Participantsgave writteninformedconsent andthisstudywasapprovedbythe University of Maryland institutional review board A total of 148 participants of an ongoing study were in cluded consisting of 69 persons with schizophrenia includ ing 15 individuals with schizoaffective disorder and 79 com munity controls all of whom completed the behavioral task forstresschallenge Behavioraldataontheinitial108partici pantsofthecurrentstudyhavebeenpresentedpreviously 25 SalivaryKYNAwasassayedin64patientsand64controls fre quency matched for age sex and smoking status These 128 participants were selected based only on these demographic characteristics ie withoutknowledgeofbehavioralresultsand priortothesalivaryassay Demographiccharacteristicsofthe samples are shown in the Table OfthepatientswithschizophreniatestedforsalivaryKYNA 11werenottakinganymedicationfor1monthorlonger 6were receiving first generation antipsychotic medications and the remaining 47 were receiving second generation antipsy chotic medications including 11 receiving clozapine and 8 re ceiving 2 or more antipsychotic medications Psychiatricsymptomswereassessedwiththe20 itemBrief Psychiatric Rating Scale total score 26and cognitive capaci ties were assessed by processing speed digit symbol coding subtestoftheWechslerAdultIntelligenceScale thirdedition 27 and working memory digit sequencing task 28Processing speed is considered the most robust cognitive domain deficit in schizophrenia 29 30 PsychologicalStressChallengeTask The stressor used for this study was developed to examine distress intolerance defined as an inability to persist in goal directed behavior while distressed 31This was operational ized using the experimental procedure illustrated in Figure 1 All participants completed a psychological stress challenge consisting of 2 computer tasks the Paced Auditory Serial Addition Task32and the Mirror Tracing Persistence Task 33 The tasks were designed to be arbitrarily difficult and puni tive to induce psychological stress to the extent that some participants would choose to forego the reward for success and quit The Paced Auditory Serial Addition Task requires participants to sum consecutive numbers presented briefly on the computer screen and to respond by using the mouse to click on the correct answer Incorrect answers cause the Research OriginalInvestigationStress InducedKynurenicAcid E2JAMAPsychiatryPublishedonlineMay 7 Copyright 2014 American Medical Association All rights reserved Downloaded From by a Yale University User on 05 16 2014 Copyright 2014 American Medical Association All rights reserved computer to play a loud 90 to 95 dB sound pressure level explosion noise The computer program tracks participant skill and titrates the speed of the presentation of the numbers to ensure that the task remains challenging despite variability in the participants cognitive abilities The Paced Auditory Serial Addition Task could last up to 12 minutes including a training session The Mirror Tracing Persistence Task asks participants to use the mouse to trace a dot along the outline of a star on the computer screen Tracing is challenging because the cursor movement is opposite to the movement of the mouse simi lar to tracing a mirror image The width of the star outline is titrateddependingonperformancetomakethechallengesimi larlydifficultacrossparticipants Anyerror touchingortrac ing outside the outline or delay in moving triggers the loud explosionnoiseandforcestheparticipanttorestartatthebe ginning The Mirror Tracing Persistence Task could last up to 13 minutes including a training session Combining the 2 tasks allows for a more rigorous defini tion of distress intolerance by reducing the potential skill dependent bias that would result if some participants were particularly skilled at arithmetic or eye hand coordination The order of tasks was randomized across participants Figure 1 After completion of the first task the participants completed a self rating of emotions see later and then immediately began reviewing instructions for the next task all under a computerized automatic sequence Participants were informed that they would receive a monetary reward based on completing the tasks but were not told the maxi mum amount of time allowed Participants were given the operational definition of distress intolerant if they quit both tasks before completion They were considered distress toler ant if they tolerated 1 or 2 tasks A monetary bonus of 20 was given for not quitting and 10 was given if the participant quit any or both tasks although this was revealed only after completion of testing After completion participants were debriefed this consisted of disclosing that the task was meant to be stressful and giving the participant an opportunity to ask questions Participants completed the Positive and Negative Affect Schedule34at baseline and after each of the 2 tasks to mea suresubjectiveaffectreactivity Figure1 Thisscaleaskspar ticipants to rate how they feel along a 5 point Likert scale for 10 positive affect and 10 negative affect NA items In this study we used only NA scores This score represents a gen eral dimension of subjective distress 34Negative affect reac tivity was defined as the maximum change in NA after com pletingeithertaskcomparedwithbaseline anditwasusedto examine whether the KYNA response if any was associated with a subjective change in NA Table DemographicCharacteristicsofParticipantsoftheEntireSampleandtheKYNAAssaySample Characteristic Entire SampleKYNA Assay Samplea Healthy Controls n 79 Patients With Schizophrenia n 69 F or 2P Value Healthy Controls n 64 Patients With Schizophrenia n 64 F or 2P Value Age mean SEM y38 9 12 8 38 1 12 4 0 17b 6838 9 12 9 37 7 12 4 0 29b 59 Male No 42 53 2 47 68 1 3 43c 0635 54 7 42 65 6 1 60c 21 Smoker No 28 35 4 33 47 8 2 33c 1422 34 4 30 46 9 2 07c 15 Working memory normalized z score mean SEM 0 25 0 89 0 35 1 06 13 6b 0010 25 0 91 0 35 1 05 11 5b 001 Processing speed normalized z score mean SEM 0 43 1 35 0 40 0 68 20 7b 0010 25 0 57 0 40 0 67 33 1b 001 BPRS total score mean SEM d 2 0 0 52 2 0 0 52 Abbreviations BPRS BriefPsychiatricRatingScale KYNA kynurenicacid aAllparticipantsintheKYNAassaysamplewereintheoverallsamplebutwere preselectedtoachievebettermatchinage sex andsmokingstatus bValuesareFscores cValuesare 2 dTheBPRSscoreswerethetotalscoresdividedbythenumberofitems 1indicatesnosymptomsinanyitems Figure1 SchematicIllustrationoftheExperimentalProcedure 40 min after testing20 min after testingSaliva collection at baseline MTPT Stress PASATQuestions on negative affect ThestresschallengeconsistedofthePacedAuditorySerialAdditionTask PASAT andtheMirror TracingPersistenceTask MTPT Theorderofthetasks wasrandomizedacrossparticipants Subjectivefeelingofnegativeaffectwas measuredusingthePositiveandNegativeAffectScheduleat3pointsduring thestudysession arrowheads Salivawascollectedatbaselineandthenagain at20and40minutesaftertheendofthestresschallenge Thedurationofthe stresschallengevariedbetweenparticipantsdependingonwhethera participantquit2 1 or0tasks Stress InducedKynurenicAcidOriginalInvestigation Research JAMAPsychiatryPublishedonlineMay 7 2014E3 Copyright 2014 American Medical Association All rights reserved Downloaded From by a Yale University User on 05 16 2014 Copyright 2014 American Medical Association All rights reserved SalivaCollectionandProcessing Saliva samples were collected at 3 times at baseline before participantsstartedeithertask andat20minutesandthen40 minutes following the completion or quitting of the second task To minimize variability in salivary KYNA levels all par ticipants were instructed to avoid eating smoking or brush ingtheirteethinthehourbeforethedistresstask andallpar ticipantswereaskedtorinsetheirmouthwithwater10minutes prior to the first saliva collection Salivawascollectedintocryovialsthroughpassivedrool and the cryovials were stored at 80 C The volume of saliva and the time used to collect each saliva sample were re corded Onthedayoftheassays salivasampleswerethawed and centrifuged 10 000g for 10 minutes and 50 L of saliva was mixed with 50 L of ultrapure water and then acidified with25 Lof6 perchloricacid Aftercentrifugation 10 000g for10minutes 20 Lofthesupernatantwassubjectedtohigh performanceliquidchromatography andKYNAwasisocrati cally eluted and fluorimetrically detected as previously described 35Assays were performed by investigators blinded to group or behavior information Intra assay reliability was assessedusingtriplicates Cortisolwasassayedinthesamesa liva samples using a commercial kit Salimetrics LLC StatisticalAnalysis Biochemical data are presented as mean SEM Repeated measures analysis of variance ANOVA was used to examine effects of diagnosis patients or controls distress intoler ance yes or no and time on salivary KYNA levels Statistical significanceusedGreenhouse Geissercorrectionswhenthere weremorethan2levelsofrepeatedmeasure Groupcategori calcomparisonsused2 sided 2tests Nonparametrictestswere used to examine group differences and correlations for de pendent variables that did not exhibit normal distributions All comparisons were 2 tailed Results BehavioralDifferencesinDistressIntolerance Patients were significantly more likely to be distress intoler ant than controls 2 8 55 P 003 Figure 2 Among pa tients those identified as distress intolerant had lower pro cessing speed than those who were not distress intolerant t62 9 4 29 P 001 buttheydidnotdifferinworkingmemory performance P 10 or Brief Psychiatric Rating Scale total score t 0 25 Controls did not differ in processing speed P 69 or working memory P 41 based on distress intol erance classification SalivaryKYNALevels InexcellentagreementwithfindingsbyKucetal 36themean SEM basal salivary KYNA level in the control group was 6 02nM 0 74nM Inpatients themean SEM levelwas7 40nM 1 05nM Figure3andeTableinSupplement Theaveragein tra assay coefficient of variation was 5 1 Repeated measuresANOVAoftime baseline and20min utes and 40 minutes after test and diagnosis patients or con trols revealedamaineffectoftime F1 4 150 9 6 01 P 007 but nosignificantinteractionbetweentimeanddiagnosis P 56 ormaineffectofdiagnosis P 84 Posthoctestsrevealedthat KYNAlevelsweresignificantlyhigherat20minutesafterthetest mean SEM 8 43nM 1 05nM thanatbaseline mean SEM 6 72nM 0 65nM P 007 At40minutesaftertest levelshad declined mean SEM 7 22nM 0 74nM but were still el evatedcomparedwithbaseline P 03 Theseresultsdemon strate a transient increase in salivary KYNA following a stress ful task that was similar in patients with schizophrenia and healthy controls when not accounting for distress intolerance Becauseofthepossibilitythatstresscancause drymouth andthusconcentratesalivaryanalytes wecalculatedaproxy measure of salivary flow rate based on the volume and time of collection for each sample Salivary flow rate actually in creased between baseline and the poststress periods in the combined sample F1 73 117 3 10 58 P 001 Thus the in creaseinsalivaryKYNAconcentrationfollowingstresswasnot a result of a reduction in saliva volume This change of sali vary flow rate did not differ between patients and controls P 31 or between distress intolerant and distress tolerant participants P 24 DistressIntoleranceandKYNAInteraction To account for behavioral differences between groups a re peated measureANOVAtestedtheKYNAresponsewheretime baselineand20minutesaftertest wastherepeatedmeasure and diagnosis patients or controls and distress response yes or no were 2 factors This analysis revealed a significant main effectoftime F1 111 5 84 P 02 andadiagnosis distressre sponse interaction