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ADORA2A Gene variation, caffeine, and emotional processing: a multi-level interaction on startle reflex.



Key Questions Addressed
1 For [population], is caffeine intake above [exposure dose], compared to intakes [exposure dose] or less, associated with adverse effects on behavior*?
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TitleData
Title ADORA2A Gene variation, caffeine, and emotional processing: a multi-level interaction on startle reflex.
Author K Domschke,A Gajewska,B Winter,MJ Herrmann,B Warrings,A Mühlberger,K Wosnitza,E Glotzbach,A Conzelmann,A Dlugos,M Fobker,C Jacob,V Arolt,A Reif,P Pauli,P Zwanzger,J Deckert,
Country
Year 2012
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Extraction Form: Behavior - Design Details - INCLUDED Studies
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Design Details
Question... Follow Up Answer Follow-up Answer
Refid 22012471
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What outcome is being evaluated in this paper? Behavior
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What is the objective of the study (as reported by the authors)? Thus, in the present double-blind, placebo-controlled study we attempted to paradigmatically investigate a multi-level pathogenetic model of anxiety by testing the effect of 300 mg caffeine citrate as an antagonist at the adenosine A2A receptor vs placebo on the emotion-potentiated (unpleasant, neutral, and pleasant International Affective Picture System pictures) startle reflex in 110 healthy individuals (male=56, female=54) stratified for the adenosine A2A receptor (ADORA2A) 1976T4 > C polymorphism (rs5751876).
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Provide a general description of the methods as reported by the authors. Information should be extracted based on relevance to the SR (i.e., caffeine related methods) A sample of 126 (male = 60, female = 66; mean age: 26.01 years, SD: 5.75) unrelated healthy subjects was consecutively recruited at the Department of Psychiatry, University of Muenster, and Department of Psychiatry, University of Wuerzburg, Germany, between 2009 and 2010. The subjects were asked to refrain from caffeine or tea consumption for 1 week prior to caffeine intervention and not to smoke, consume alcohol (assessed by a breath test), or take any medication for at least 24 h prior to the investigation. Subjects were genotyped for the ADORA2A 1976T4>C (rs5751876) polymorphism according to published protocols (see Alsene et al, 2003; Deckert et al, 1998). According to previous findings, subjects were stratified into risk (TT) and non-risk (CT/CC) genotype carriers (cf. Alsene et al, 2003; Childs et al, 2008; Deckert et al, 1998; Rogers et al, 2010). The study used a one-session, double-blind, placebo-controlled, between-subject design. Caffeine intervention was performed by oral administration of 300 mg caffeine citrate (Fagron, Barsbuettel, Germany; equivalent to 150 mg freebase caffeine), which has been shown to be close to the threshold for producing anxiogenic effects and as such might be the optimal dose to detect subtle genotype effects (cf. Alsene et al, 2003; Childs et al, 2008; Rogers et al, 2010). Caffeine citrate was administered in white opaque gelatin capsules; placebo capsules contained mannitol and aerosil (99.5 : 0.5) according to the German Drug Law (Arzneimittelgesetz; AMG). At about 0900 hours - after the first self-report anxiety measurement - subjects were administered a placebo or caffeine capsule with a glass of water. Twenty-four emotionally threatening unpleasant images taken from the International Affective Picture System (IAPS; Lang et al, 2005) were selected as anxiety-relevant emotional cues along with 24 neutral and 24 pleasant IAPS pictures. Ninety-five percent of all pictures were exactly the same for both genders, whereas different erotic pictures were chosen for men and women to ensure comparable valence and arousal levels. At the assumed maximum plasma level of caffeine and the described time to peak increases in self-report ratings of anxiety, respectively (60 min after administration; Alsene et al, 2003; Childs et al, 2008; Rogers et al, 2010), the emotion-potentiated startle experiment was started (at 1000 hours). In order to get subjects used to the startle stimulus (50 ms of 95-dB white noise with an instantaneous rise-time presented through Bose Around-Ear Headphones) and to prevent outlier startle responses during the critical trials, eight startle stimuli at random intervals of 1–12 s were presented. The startle experiment per se consisted of three blocks of 24 anxiety-relevant, neutral, or pleasant IAPS pictures, respectively, as described above, and 3-min breaks between the blocks. An experimental block contained eight pictures of each of the three categories in random order, with the constraint that no two of the same type (unpleasant, neutral, or pleasant) were presented successively. Electromyogram activity of the M. orbicularis oculi, which is responsible for eyelid closure, was measured as a commonly used variable for recording startle reactions in humans (Blumenthal et al, 2005). Startle data were checked for zero responses and artifacts in each subject. Startle reactions with no detectable responses (o5 mV) were scored as 0. Artifacts were defined as spontaneous eye blinks during baseline or within 20 ms after startle probe onset, and were scored as missing values. Subjects were excluded from data analysis when having too many zero responses (more than 2.5 SDs above mean number of zero responses) or less than three valid startle responses in one picture category. All startle magnitudes were T-transformed within subjects in order to assure comparability of data (for methodical overview Blumenthal et al, 2005; Muhlberger et al, 2008; Pauli et al, 2010). Subjects were asked to rate their anxiety level by placing a mark on a visual analogue scale (VAS) consisting of a 100 mm horizontal line labeled ‘not at all anxious’ (score of 0) and ‘extremely anxious’ (score of 100). Additionally, the 35-item German version of the Profile of Mood States (POMS) was administered, on which subjects report their current mood on a seven-point scale from ‘not at all’ (score of 0) to ‘extremely’ (score of 6) (Biehl et al, 1986; McNair et al, 1992). Four categories have been distinguished within the German version of the POMS: (1) ‘Depression–Anxiety,’ (2) ‘Fatigue,’ (3) ‘Vigor,’ and (4) ‘Hostility’ (cf. Albani et al, 2005). In the present study, the subscale ‘Depression– Anxiety’ was used as a subjective measure of anxiety. VAS and POMS measurements were taken at three points in the course of the study: (1) before the respective intervention (caffeine/placebo); (2) at maximum caffeine plasma level and the described time to peak increases in self-report anxiety, respectively, (60 min; Alsene et al, 2003); and (3) after emotion-potentiated startle. Sample characteristics were evaluated by x^2 tests for genotype (ADORA2A 1976TT risk vs. 1976CC/CT non-risk genotypes) and gender with intervention (caffeine versus placebo) as between-subjects-factor as well as by one-way ANOVAs for caffeine consumption, age and anxiety sensitivity with intervention, genotype, and gender as between-subjects-factors. VAS and POMS ratings were analyzed by ANOVAs for repeated measures, with genotype and intervention (caffeine vs placebo) as between-subject factors, and measurement time (three measurement times: before substance intake, 1 h after substance intake, and after the startle experiment) as a within-subject factor. Baseline startle response (assessed in the ITIs) was analyzed by ANOVA for repeated measures, with measurement time (the 12 ITI startle responses were divided into four measurement times (T1–T4) each being the mean of three consecutive startle responses) as a within-subject factor and genotype and intervention as between-subject factors. Picture ratings, which were conducted at the end of the startle experiment (see Figure 1), and picture viewing times - defined as time periods between picture onset and consecutive ratings - were analyzed by ANOVA for repeated measures, with genotype and intervention as between-subject factors, and picture category (unpleasant, neutral, and pleasant) as a within-subject factor. Pairwise comparisons of picture valence or time of measurement were assessed by post-hoc t-tests. According to a priori hypotheses, the main multi-level analysis of emotion-potentiated startle response was performed by ANOVA for repeated measures, with genotype and intervention as between-subject factors, and picture category (unpleasant, neutral, and pleasant) as a within-subject factor. Picture categories were further analyzed by post-hoc univariate ANOVAs. Further explorative analyses were performed by using gender and AS (median split) as additional factors for all above mentioned ANOVAs. Alpha level was set at 5% using Greenhouse–Geisser corrections where appropriate.
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How many outcome-specific endpoints are evaluated? 1
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What is the (or one of the) endpoint(s) evaluated? (Each endpoint listed separately) Anxiety
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List additional health endpoints (separately).
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List additional health endpoints (separately)
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Notes Anxiety was assessed by multiple measures for various genotypes of the sample population.
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Clinical
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Physiological
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Other Other
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What is the study design? Controlled Trial
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Randomized or Non-Randomized? NCT
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What were the diagnostics or methods used to measure the outcome? Both
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Optional: Name of Method or short description International Affective Picture System; Emotion-Potentiated Startle Paradigm; Visual Analog Scale; POMS mood questionnaire
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Caffeine (general)
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Coffee
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Chocolate
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Energy drinks
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Gum
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Medicine/Supplement
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Soda
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Tea
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Measured Measured
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Self-report
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Children
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Adolescents
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Adults Adults
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Pregnant Women
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What was the reference, comparison, or control group(s)? (e.g. high vs low consumption, number of cups, etc.) Placebo vs caffeine (300 mg caffeine citrate; equivalent to 150 mg caffeine)
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What were the listed confounders or modifying factors as stated by the authors? (e.g. multi-variable components of models.  Copy from methods) Sample characteristics were evaluated by X^2 tests for genotype (ADORA2A 1976TT risk vs. 1976CC/CT non-risk genotypes) and gender with intervention (caffeine versus placebo) as between-subjects-factor as well as by one-way ANOVAs for caffeine consumption, age and anxiety sensitivity with intervention, genotype, and gender as between-subjects-factors.
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Provide a general description of results (as reported by the authors). There was no significant main effect of intervention (caffeine vs placebo) on mean startle magnitudes (F(1, 106) = 0.43; p = 0.51). Also, no significant interaction effect of intervention (caffeine vs placebo) and picture category was observed (F(2, 212) = 0.04, p = 0.95). A significant main effect of measurement time on VAS anxiety ratings was observed (F(2, 212) = 11.41, p < 0.001), with significantly decreasing anxiety levels from point-1 (before capsule intake) to point-2 (1 h after capsule intake; t(109) = 2.76, p = 0.007) and significantly increasing anxiety levels from point-2 to point-3 (after the startle experiment; t(109) = 4.51, p<0.001) (Figure 2). However, there were no significant effects of genotype, intervention, or genotype intervention on VAS ratings (all F(2, 212)<1.28, p>0.28). Analysis of the POMS subscale ‘Depression–Anxiety’ revealed a significant main effect of measurement time (F(2, 212) = 22.52, p<0.001), with highest scores at the end of the experiment and lowest scores prior to the startle experiment (point-3>point-2 and point-1; point-2 < point- 1; all t(109)> |2.76|, p<0.008). There were no significant effects of genotype or intervention (both F(2, 212)<1.30, p>0.27), but a significant interaction effect of genotype X intervention (F(2, 212) = 5.25, p = 0.02). Further analysis revealed that the above mentioned pattern (point-1>point- 2<point-3) was valid for the ADORA2A non-risk genotype group under both conditions and for the risk genotype group only under verum (all t(26)>|2.26|, p<0.03), but not under placebo (all t(25)<|1.75|, p>0.09). In risk genotype carriers, increase in POMS ‘Depression–Anxiety’ ratings from point-2 to point-3 was significant in the verum condition (t (26) = -5.20, p<0.001) but not in the placebo condition (t (25) = 1.10, p = 0.28).
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Did the authors perform a dose-response analysis (or trend/related analysis)? No
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What were the authors's observations re: trend analysis?
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What were the author's conclusions? A synergistic effect of the ADORA2A risk genotype and caffeine on startle response to negative emotional stimuli, in ADORA2A 1976TT risk genotype carriers highest startle magnitudes were observed after caffeine administration in response to unpleasant emotional material, with caffeine now eliciting a significant contrast between unpleasant and neutral emotional stimuli. Reciprocally, ADORA2A 1976CC/CT low-risk genotype carriers challenged with caffeine show equally high startle magnitudes for unpleasant and neutral pictures, whereas under placebo - as expected in an unmodulated emotion-potentiated startle paradigm in healthy probands - a gradual increase in startle response from pleasant to neutral to unpleasant emotional material was observed. Thus, subjects carrying either the ADORA2A anxiety 1976TT risk genotype or receiving caffeine, respectively, as single risk-increasing factors react to unpleasant and neutral stimuli in an equally aroused manner as reflected by comparable startle magnitudes, whereas in a multi-level risk model caffeine in synergy with genotype further increases startle reaction specifically for unpleasant emotional stimuli. We did not discern a significant influence of caffeine (150 mg freebase) on VAS or POMS measures of anxiety as opposed to previous studies (Alsene et al, 2003; Childs et al, 2008). There might be several reasons for that: First, the presently applied 35-item German version of the POMS scale is not fully comparable to the American version (65 items; Albani et al, 2005; McNair et al, 1992). A lower than average mean AS in the present sample (cf. Peterson and Reiss, 1992) might further account for low anxiety ratings on VAS or POMS, respectively. Also, the presently administered dose of caffeine might have been too low to elicit increased self-report anxiety, as Childs et al (2008) observed increases in VAS anxiety ratings only after administration of 450 mg caffeine, but not at the equivalent of 150 mg freebase caffeine, and other studies have shown that only high doses of caffeine increase anxiety (cf. Evans and Griffiths, 1991; Griffiths and Woodson, 1988). Finally, the lack of a cross-over design in the present study might have prevented detection of effects on self-report data.
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What were the sources of funding? This study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; SFB-TRR-58 project C2 to KD and JD; project Z2 to JD, AR, and PP; project B1 to PP and AM; and project C1 to PZ). We acknowledge the technical support by Max Hilscher, Heike Ewald, Thilo Rattay, and Julian Conrad.
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What conflicts of interest were reported? All affiliations mentioned below have no relevance to the work covered in the manuscript: AR has received a research grant from Astra Zeneca. KD has received speaker fees from Pfizer, Lilly, and Bristol-Myers Squibb; she is a consultant for Johnson & Johnson and has received funding from Astra Zeneca. In the past 3 years, JD has received speaker’s honoraria by Janssen, Bristol-Myers Squibb, Wyeth, Lundbeck, Astra Zeneca, and Pfizer, and grant support from Medice. PZ has received speaker fees from Pfizer, Servier, Lilly, Astra Zeneca, and Bristol-Myers Squibb; he is on the advisory board of Pfizer, is a consultant for Ironwood Pharmaceuticals, and has received funding from AstraZeneca. VA is member of advisory boards and/or gave presentations for the following companies: Astra-Zeneca, Janssen-Organon, Lilly, Lundbeck, Servier, Pfizer, and Wyeth. He chaired the committee for the ‘Wyeth Research Award Depression and Anxiety’. All other authors have no conflicts of interest to declare, financial or otherwise.
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Does the exposure (dose) need to be standardized to the SR? No
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Provide calculations/conversions for the exposure based on the decision tree in the guide (for all endpoints/exposure levels of interest).
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List all the endpoint(s) followed by the dose (mg) which will be used in comparison to Nawrot.  Characterize value as LOAEL/NOAEL, etc. if possible.  Anxiety NOAEL = 150 mg caffeine (total study population) Anxiety LOAEL = 150 mg caffeine (ADORA2A TT genotype only)
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Notes regarding selection/listing of endpoints and exposures/doses to be compared to Nawrot. The anxiety NOAEL was determined for the combined sample population when comparing placebo to caffeine interventions. a LOAEL was reported for the 'Depression-Anxiety' subscale ratings of the POMS in the 'risk genotype' group but not the 'non-risk genotype' group. This study only involved one exposure level for caffeine.
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What is the importance of the study with respect to the adverseness of the outcome? Important
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