The acute effects of l-theanine in comparison with alprazolam on anticipatory anxiety in humans

Hum Psychopharmacol Clin Exp 2004; 19: 457-465.
Published online 26 July 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/hup.611 The acute effects of L-theanine in comparison withalprazolam on anticipatory anxiety in humans Kristy Lu1, Marcus A. Gray1, Chris Oliver2, David T. Liley1, Ben J. Harrison1,Cali F. Bartholomeusz1, K. Luan Phan3 and Pradeep J. Nathan1* 1Neuropsychopharmacology Laboratory, Brain Sciences Institute, Swinburne, University of Technology, Victoria, Australia2Blackmore's Ltd, NSW, Australia3Department of Psychiatry and Behavioural Neuroscience, Wayne State University, Detroit, MI, USA L-Theanine (-glutamylethylamide) is one of the predominant amino acids ordinarily found in green tea, and historically hasbeen used as a relaxing agent. The current study examined the acute effects of L-theanine in comparison with a standardbenzodiazepine anxiolytic, alprazolam and placebo on behavioural measures of anxiety in healthy human subjects using themodel of anticipatory anxiety (AA). Sixteen healthy volunteers received alprazolam (1 mg), L-theanine (200 mg) or placeboin a double-blind placebo-controlled repeated measures design. The acute effects of alprazolam and L-theanine wereassessed under a relaxed and experimentally induced anxiety condition. Subjective self-reports of anxiety including BAI,VAMS, STAI state anxiety, were obtained during both task conditions at pre- and post-drug administrations. The resultsshowed some evidence for relaxing effects of L-theanine during the baseline condition on the tranquil-troubled subscaleof the VAMS. Alprazolam did not exert any anxiolytic effects in comparison with the placebo on any of the measures duringthe relaxed state. Neither L-theanine nor alprazalam had any significant anxiolytic effects during the experimentally inducedanxiety state. The findings suggest that while L-theanine may have some relaxing effects under resting conditions, neither L-theanine not alprazolam demonstrate any acute anxiolytic effects under conditions of increased anxiety in the AA model.
Copyright # 2004 John Wiley & Sons, Ltd.
key words - L-theanine; alprazolam; anticipatory anxiety; anxiety; GABA; anxiolytic; anxiety models; benzodiazepine bition of the glutamate transporter (Sadzuka et al.,2001); (2) increases in -aminobutyric acid (GABA) L-Theanine (-glutamylethylamide) is one of the pre- concentrations (Kimura and Murata, 1971); (3) dominant amino acids found in green tea and histori- increases in dopamine release in the striatum in rats cally it has been used as a relaxing agent. It was first (Yokogashi et al., 1998a); (4) increases in serotonin isolated and identified in green tea leaves (Camella levels in specific brain regions including the striatum, sinensis) in 1949 by Sakato (1949) and in mushrooms hippocampus and hypothalamus in rats (Yokogashi (Xerocomus badius) in the early 1950s (Casimir et al., et al., 1998b); and (5) neuroprotective effects in the hippocampus through blockade of multiple glutamate The pharmacology of L-theanine is relatively receptor subtypes, NMDA (N-methyl-D-aspartate) unknown. Animal studies have shown evidence for and AMPA (-amino-3-hydroxy-5-methylisoxazole- multiple pharmacological effects on various neuro- 4-propionic acid) receptors (Kakuda, 2002; Kakuda chemical systems. These pharmacological effects include: (1) inhibition of glutamate reuptake by inhi- While historically L-theanine has been shown to have relaxing properties (Juneja et al., 1999; Luet al., 2004), the anxiolytic effects of L-theanine have * Correspondence to: Professor P. J. Nathan, Neuropsychopharma- not been established scientifically in animal or human cology Laboratory, Brain Sciences Institute, Swinburne University studies. However, the pharmacological effects of L- of Technology, Melbourne, Australia. Tel: (03) 9214 5216. Fax: (03)9214 5525. E-mail: pnathan@bsi.swin.edu.au theanine reported in animals suggest that it may have Copyright # 2004 John Wiley & Sons, Ltd.
some anxiolytic properties given that both serotonin especially panic disorder (Barlow et al., 1996). Pre- and GABA play a fundamental role in the neurobiol- viously AA has been used as a model of anxiety and ogy of anxiety and are molecular targets in the treat- is induced within healthy human subjects via the ment of various anxiety disorders (Kent et al., 2002; expectation of mild electric shocks (Reiman et al., Charney, 2003; Millan, 2003). Supporting the pre- 1989; Chua et al., 1999; Simpson et al., 2001; Gray clinical pharmacological effects of L-theanine, one et al., 2003). Induced AA is associated with increased electrophysiological study in healthy human subjects subjective anxiety, phasic skin conductance and heart reported possible relaxing effects of L-theanine rates, and produces changes in blood flow and electri- (200 mg) as indicated by increased alpha activity in cal activity in cortical areas associated with anxiety the occipital and parietal cortex (Ito et al., 1998).
(Reiman et al., 1989; Chua et al., 1999; Simpson While useful, the latter finding does not provide strong et al., 2001; Gray et al., 2002, 2003). Recently, in evidence for an anxiolytic effect of L-theanine, as an electrophysiological brain imaging study it was alpha activity is regarded as an indirect and crude shown that this model of anxiety is also sensitive to measure anxiety and behavioural measures of anxiety the three main classes of anxiolytics, namely selective or relaxation were not evaluated and reported.
serotonin-reuptake inhibitors (SSRI) (citalopram), 5- Both pharmacological and psychological methods HT1A partial agonists and benzodiazepines (alprazo- have been used to examine anxiety in humans. These include pharmacological methods such as cholecysto- Given the sensitivity of the AA model to anxioly- kinin tetrapeptide (CCK-4), lactate, carbon dioxide tics, the acute effects of L-theanine were examined and pentagastrin induced anxiety (Benkelfat et al., in comparison with a standard benzodiazepine anxio- 1995; Bellodi et al., 1998; Ponto et al., 2002; lytic, alprazolam, on behavioural measures of anxiety Bradwejn et al., 1995; Javanmard et al., 1999; in healthy human subjects using the AA model. It was Boshuisen et al., 2002; Zedkova et al., 2003; Zwanz- hypothesized both alprazolam and L-theanine would ger et al., 2003a, 2003b), and psychological (experi- reduce the subjective experience of anxiety in the mental) methods such as extemporaneous public AA model, and that the effects of L-theanine would speaking, aversive conditioning, fear-potentiated star- be comparable to that of alprazolam.
tle response, Stroop colour word task performanceand anticipation of electric shock (Baas et al., 2002;Chua et al., 1999; Graeff, 2002, 2003; Grillon and Ameli, 2001; Grillon et al., 1991, 1993a,b; Palmaet al., 1994; Reiman et al., 1989; Riba et al., 2001; Silva et al., 2001; Simpson et al., 2001; Tillfors Sixteen healthy participants (12 males aged between et al., 2002). Recently, it has been shown that some 18 and 34 years (mean Æ SD ¼ 24.8 Æ 5.4) and four of these models of experimental anxiety, particularly females aged between 28 to 31 years (mean Æ the fear-potentiated startle response are also sensitive SD ¼ 29.0 Æ 1.4)) were recruited through University to benzodiazepine anxiolytic agents (Zuardi et al., advertisements. All participants were considered for 1993; Patrick et al., 1996; Hellewell et al., 1999; Leite selection if they were healthy, non-smokers, non- et al., 1999; Bitsios et al., 1999; Riba et al., 2001; medicated (no contraceptive medication for females), Graeff, 2003). Furthermore, the 5-HT agonist (d- and had no known personal or family history of fenfluramine), and 5-HT antagonist (nefazodone) have physical or psychiatric disorders as determined by also been shown to attenuate the anxiety induced by semi-structured clinical interview by a physician.
both simulated public speaking and aversive condi- All participants gave written informed consent to the tioning to tones, as indicated by the anxiety dimension take part in the study, which was approved by the of the VAMS and the bodily symptoms scale (BSS) Swinburne Research Ethics Committee.
(Hetem et al., 1996; Silva et al., 2001; Graeff 2002,2003). These findings suggest that experimentalmodels of anxiety may be useful in detecting acute anxiolytic effects of potential anxiolytic drugs.
Anticipatory anxiety (AA) is one of the basic forms The study was a double-blind, placebo-controlled, of anxiety, and commonly occurs in response to an repeated measures design, in which all subjects were immediate negative event or stressor (Reiman et al., tested under three treatment conditions. The treatment 1989). It is commonly experienced in normal indivi- duals and in patients suffering from anxiety disorders, Suntheanine1, Taiyo Kagaku, Japan) and alprazolam Copyright # 2004 John Wiley & Sons, Ltd.
Hum Psychopharmacol Clin Exp 2004; 19: 457-465.
Subject testing schedule and behavioural measures of anxiety in each treatment day In an attempt to control for metabolic differences, Alprazolam was used as a positive control in order subjects were instructed to consume a light breakfast to compare its effects with L-theanine. Individual with a low protein content such as toast or cereal prior assignment to the order of treatment condition was to each testing day. They were instructed not to con- randomized using a Latin square design. All partici- sume alcoholic or caffeinated beverages, including pants were required to attend 3 full-day repeated test- coffee or tea, particularly green tea, in the previous ing sessions with a minimum 7 days between testing 24 h. A standard meal (one apple and 300 ml of orange days to allow for a sufficient drug washout period. On juice) was provided 3½ h after drug administration.
each treatment session, testing was conducted at base- Female participants were tested during the follicular line (pre-treatment) and 2½ h and 5 h post-treatment phase (days 1-13) of their menstrual cycle in order (see Figure 1). Testing times were selected to coincide to control for the possible influence of phase-depen- with peak pharmacokinetic and pharmacodynamic effects of alprazolam and L-theanine (Fawcett and Figure 1 shows the subjects' testing schedule on Kravitz, 1982; Terashima et al., 1999). A total of nine each treatment day. At baseline (pre-drug testing), testing sessions was completed in the 3 day testing the subjects were asked to complete pre-anxiety self-rating measures including the Beck depressioninventory-II (BDI-II) and trait and state anxiety (seeBehavioural Measures Section). Subjects were then asked to complete the AA task conditions (see Testing The study was conducted at the Neuropsychopharma- Methodology section). Behavioural measures of anxi- cology Laboratory, Brain Sciences Institute, Swin- ety including Beck anxiety inventory (BAI) and burne University of Technology. Subjects were VAMS were used to measure subjective anxiety dur- initially interviewed by telephone to screen for health ing the AA task. Subjects received either L-theanine and fitness suitability. All subjects were screened (200 mg), alprazolam (1 mg) or placebo immediately further for psychiatric and physical illnesses by a after the baseline testing was completed. The AA task medical physician using the Prime-MD (based on and behavioural measures of anxiety were re-adminis- the DSM-IV criteria for psychiatric disorders) (APA, tered at 2½ and 5 h post-drug administration. A side- 1994) and a semi-structured clinical interview. At the effect checklist was also administered to monitor time of the initial telephone contact, subjects were subjective physiological symptoms. The checklist given a verbal explanation of the study. Upon their took the form of a 1-5 Likert scale, that contained the arrival in the laboratory for the medical examination, following items: headache, feeling cold, feeling hot, written instructions and information about the study dizziness, blurred vision, nausea, heart palpitations, was provided and they were asked to sign the consent dry mouth and gastric complaints, to give a mean sub- form if they agreed to take part in the study.
jective physiological symptoms score for each item.
Copyright # 2004 John Wiley & Sons, Ltd.
Hum Psychopharmacol Clin Exp 2004; 19: 457-465.
Testing methodology (anticipatory anxiety) The Beck depression inventory-II (BDI-II: Beck et al., 1996) is a widely used 21-item inventory for The subjects completed two task conditions, an AA assessing the severity of depression. Each item is rated condition and a relaxed condition. In both conditions, on a scale ranging from 0 'normal' to 3 'most severe' the subjects were instructed to focus their gaze on a with summary scores ranging from 0 to 63.
computer monitor and focus their attention on their The Beck anxiety inventory (BAI: Beck and Steer, current feelings. During the AA condition, a red bor- 1987) consists of 21 items assessing anxiety symp- der framed the computer screen. Subjects were toms, especially focuses on those symptoms that are informed that they would randomly receive electrical distinct from depressive symptoms. Each item is rated shocks during the red border presentation. Subjects on a 4-point scale ranging from 0 'not at all' to 3 completed two blocks of AA tasks in this condition, 'severely, I could barely stand it', with a total score which had a total of 180 s duration, 135 s on the first block and 45 s on the second block. The subjects alsostopped to complete the VAMS and Beck anxietyinventory (BAI) measures in between the two blocks (see Behavioural Measures). The relaxed (baseline) The data were analysed using the Statistical Package condition was identical to the AA condition, except for the Social Sciences (SPSS for Windows, version in this condition, a blue border framed the computer 11, SPSS Inc., Chicago II USA). The maximum screen and subjects were informed that no shocks effects of each treatment on anxiety were calculated would be delivered during the blue border presenta- as the difference between the mean scores at post drug tion. The current experimental methodology is similar (maximum value regardless of time, i.e. 2½ or 5 h) to that used in previous neuroimaging studies and has relative to the baseline mean score and the baseline been shown to activate areas associated with anxiety alprazolam, placebo) and time (baseline testing,post-treatment maximum score) were the independent Stimuli and apparatus. The electrical stimuli were variables, and behavioural measures (VAMS sub- delivered through two electrodes with gel and adhe- scales, BAI and STAI) were the dependent variables.
sive attached to the back of the participant's right The data were analysed using a 3 (drug) by 2 (time) hand. The stimulus was delivered by an Isolated Sti- repeated measures multivariate analysis of variance mulator, Dogwood Scientific Equipment, model (MANOVA), conducted separately for both the CMS 1-200. The shocks had an intensity of 30 mA, relaxed (baseline) and AA task conditions. Post hoc a voltage of 110 V (maximum) and duration of analyses were carried out on significant interactions 0.1 ms. This level of electrical stimulation has pre- between drug and time in order to examine the speci- viously been shown to reliably induce anticipatory fic effects of each drug. STAI Trait anxiety was also anxiety in healthy human subjects without causing used as a covariate in a multivariate analysis of covar- iance (MANCOVA), in order to examine the influenceof trait anxiety on the drug induced changes in beha-vioural anxiety in the AA model. In addition, to exam- ine the effects of the task conditions (relaxed vs AA) The following self-rating scales were used to assess on measures of anxiety, a paired-sample t-test was behavioural (subjective) states of anxiety.
conducted for each behavioural anxiety measure using The visual analogue mood scale (VAMS: Bond and Lader, 1974) requires subjects to place a single markwith a pen along a 100 cm horizontal line separated by two adjectives in the current study: (1) calm-excited,(2) relaxed-tense and (3) tranquil-troubled.
The BDI scores (M ¼ 3.81, SD ¼ 4.78) and trait anxi- The state-trait anxiety inventory (STAI: Spielberger ety scores (M ¼ 33.44, SD ¼ 7.14) indicated that all et al., 1970) is a 20-item scale assessing two types of subjects' scores were within the normal range.
anxiety: state anxiety measures the intensity of anxi- Paired-sample t-test showed that all behavioural ety at a particular moment from 1 'not at all' to 4 'very measures were associated with increased subjective much so', and trait anxiety measures anxiety as a rela- anxiety in the AA task condition relative to the relaxed tively stable personality trait from 1 'almost never' to Copyright # 2004 John Wiley & Sons, Ltd.
Hum Psychopharmacol Clin Exp 2004; 19: 457-465.
Mean and standard deviation (SD) for behavioural dition are shown in Table 2. Further post hoc measures of anxiety in both relaxed and AA condition comparisons were performed for both subjective mea- sures of anxiety. Alprazolam significantly increasedSTAI state anxiety scores in the relaxed condition in comparison with placebo (F(1, 15) ¼ 6.11, p < 0.05).
L-theanine had no significant effect in comparison with placebo (p> 0.05). With regard to the tranquil- troubled subscale of the VAMS, L-theanine signifi- cantly reduced subjective anxiety in comparison with alprazolam (F(1, 15) ¼ 5.37, p < 0.05) and placebo ap < 0.05 for the AA relative to relaxed condition.
(F(1, 15) ¼ 4.73, p < 0.05). In the AA condition, therepeated measures MANOVA failed to reveal any sig- subscale of in the VAMS (t(47) ¼ 2.28, p > 0.05) nificant drug  time interactions. The means and stan- showed significant differences between the two task dard deviations and the associated significant levels conditions. The means and standard deviations (SD) for the drug  time interaction on each measure of for the behavioural measures of anxiety in both anxiety in the AA condition are shown in Table 3.
relaxed and AA conditions are shown in Table 1.
The results were also re-analysed using trait anxiety In the relaxed task condition, the repeated measures scores on the STAI as a covariate in both the relaxed MANOVA revealed a significant drug  time interac- and anxious conditions. Subsequent repeated mea- tion for two of the dependent variables, STAI state sures MANCOVA failed to show any significant anxiety and the tranquil-troubled subscale of the drug  time interactions for any of the anxiety mea- VAMS. The means and standard deviations, and the sures. In addition no significant adverse effects were associated significant levels for the drug  time inter- found between the treatment conditions (i.e. L-thea- action on each measure of anxiety in the relaxed con- nine or alprazolam) in comparison with placebo.
Effects of placebo, L-theanine and alprazolam on subjective anxiety in the relaxed condition. Results are expressed as mean and standard deviation (SD). The F and p values refer to drug  time interaction for the repeated measures MANOVA n ¼ 16; ap < 0.05 for alprazolam vs placebo (Greenhouse-Geisser); bp < 0.05 for L-theanine vs placebo and L-theanine vs alprazolam.
Effects of placebo, L-theanine and alprazolam on subjective anxiety in the AA condition. Results presented as mean and standard deviation (SD). The F and p values refer to drug  time interaction for the repeated measures MANOVA Copyright # 2004 John Wiley & Sons, Ltd.
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suggesting that drugs that increase serotonin, enhanceconditioned fear responses while inhibiting uncondi- To our knowledge this is the first study to examine the tioned fear (for review see Graeff, 2002, 2003). The anxiolytic effects of L-theanine. The study examined differential effects of pro-serotonergic compounds the acute effects of L-theanine in comparison with have been hypothesized to reflect region-specific the benzodiazepine, alprazolam and placebo on beha- changes in the pre-frontal cortex versus the periaque- vioural measures of anxiety in healthy human subjects ductal grey (Deakin and Graeff, 1991). It is possible under both a relaxed (baseline) state and during an that benzodiazepines may also have region-specific experimentally induced anxiety state (i.e. anticipation effects (depending on the anxiety model or state), of a mild electric shock). The findings provide some which could account for varying behavioural changes evidence to support a relaxing effect of acute L-thea- nine administration during the relaxed (baseline) An important observation in the current study was experimental state, with subjects reporting to be more that neither L-theanine nor alprazolam demonstrated tranquil in the tranquil-troubled dimension of the any effects on subjective anxiety levels under the VAMS compared with placebo. In comparison, alpra- experimentally induced anxiety condition. While zolam did not exert any anxiolytic effects when com- the reason for the lack of effect of L-theanine during pared with placebo on any of the measures during the the experimentally induced anxiety condition (in com- relaxed state. In addition, neither L-theanine nor alpra- parison with the mild anxiolytic effects reported under zolam demonstrated significant anxiolytic effects dur- resting conditions) is not known, it is likely that the intensity of anxiety was too strong in the experimental suggesting that under conditions of increased anxiety, anxiety condition, leading to an ineffective anxiolytic neither drug had measurable anxiolytic effects.
effect. In addition, given that L-theanine only demon- The finding of some calming or relaxing effect of L- strated weak anxiolytic effects on the resting (base- theanine in the resting (baseline) state is consistent line) state, it is not surprising that no anxiolytic with a previous report indicating that such effects effects were observed under conditions of increased may correspond with increases in alpha band electro- anxiety. The absence of an anxiolytic effect of alpra- cortical activity in the occipital and parietal cortex (Ito zolam is consistent with a number of studies, which et al., 1998). Furthermore the current findings support fail to show an anxiolytic effect of benzodiazepines the historical use of green tea as a relaxing agent (for during experimentally induced anxiety (for review review see; Lu et al., 2004). However, in all cases the see; Graeff, 2003). For example, a number of benzo- evidence for this effect is not strong. For example, in diazepines have been shown to have no effects on self- our study L-theanine was only found to affect one of reported anxiety in the stimulated public speaking the anxiety subscales (i.e. tranquil-troubled subscale model (Graeff et al., 1985; McNair et al., 1982), and of the VAMS), while in the study of Ito et al.
the Stroop colour-word test (Tulen et al., 1991). How- (1998), the measure of anxiety indexed by changes ever, it should be noted that the latter studies, includ- in alpha band activity would be considered an indirect ing our current study used behavioural (subjective) and possibly a crude measure of the anxious state.
measures of anxiety and a growing body of research Surprisingly, alprazolam did not exert anxiolytic or suggests that benzodiazepines may demonstrate calming effects in the relaxed or baseline experimen- anxiolytic effects when measured objectively using tal condition. Paradoxically there was an increase in physiological measures (i.e. skin conductance, startle subjective reports of anxiety in the STAI state anxiety response, brain electrical activity). For example, a measure. While it is difficult to explain this increase in number of studies have suggested that benzodiaze- state anxiety with alprazolam, it should be noted that pines including alprazolam could inhibit the fear, there have been inconsistencies in the literature with darkness or context potentiated startle response regard to the effects of benzodiazepines on subjective (Patrick et al., 1996; Bitsios et al., 1999; Riba et al., reports of anxiety under resting conditions. Some stu- 2001) and benzodiazepines have been shown to be dies have reported an anxiolytic effect of alprazolam sensitive in a model of aversive conditioning of the (Riba et al., 2001) and also other benzodiazepines skin conductance responses to tones (Hellewell (McNair et al., 1982; Guimaraes et al., 1989), while et al., 1999). Similarly, our recent findings using SSPT other studies suggest a lack of effect of a number of suggests that the electrophysiological responses in the benzodiazepines (Baas et al., 2002) on state anxiety frontal and temporal cortex during anticipatory anxi- under resting conditions. Similar discrepancies have ety are attenuated with a number of anxiolytics been noted with serotonergic anxiolytics with findings including alprazolam following acute administration Copyright # 2004 John Wiley & Sons, Ltd.
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(Gray et al., 2002). These findings suggest that while effects. This is supported by the clinical studies that subjective measures may not be sensitive in detecting demonstrate anxiolytic effects of alprazolam in anxi- drug induced anxiolytic effects, the effects of L-thea- ety disorders (including generalized anxiety disorder nine and alprazolam may be observed or detected in and panic disorder) only following chronic adminis- models of anxiety incorporating objective physiologi- tration (Laakmann et al., 1998; Lydiard et al., 1997; cal measures of anxiety such as startle, skin conduc- Sheikh and Swales, 1999). Hence one cannot rule tance response or electrophysiological imaging. This out a possible anxiolytic effect of both L-theanine is not surprising given that the neural networks/ and alprazolam in the anticipatory anxiety model, circuits involved in the physiological measures with higher doses or following chronic administration.
(i.e. startle) may be modulated differentially by neuro- In summary, the findings of the current study sug- chemicals compared with networks involved with gest that under conditions of experimentally induced subjective self reports. For example, the startle anticipatory anxiety, neither L-theanine nor alprazo- response originates from structures located in the lam had any acute anxiolytic effects in healthy sub- brain stem and is modulated by descending pathways jects as measured by behavioural measures of (Davis, 1984), while subjective changes during antici- anxiety. However, there was some evidence for a patory anxiety are thought to involve a neuroanatomi- calming or relaxing effect of L-theanine under resting cal circuitry that includes the prefrontal, anterior conditions providing some support for the historical temporal and occipital cortex and the insula (Chua use of green tea as a relaxing agent. Further studies et al., 1999; Gray et al., 2003). Alternatively, anxioly- are warranted to examine the effects of L-theanine in tic effects may be observed in pharmacological comparison with alprazolam using objective physiolo- models of anxiety, such as the CCK-4 induced panic gical measures of anxiety. In addition, dose response model in which benzodiazepines, including alprazo- studies or chronic dosage studies are required to lam have been shown to reduce CCK-4 induced panic further examine the possible efficacy of L-theanine (de Montigny, 1989; Zwanzger et al., 2003a). The lat- ter findings may also be related to specific modulationof neural networks (within the neocortical and limbic areas, which have been shown to co-localize bothCCK and GABA) (Somogyi et al., 1984).
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