Department of Psychiatry, Manchester Royal Infirmary, Manchester
Department of Psychiatry, University of Manchester, Manchester
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* Previously presented at the Annual Meeting of the British Association for
Psychopharmacology in association with the British Neuropsychiatry
Association, 16-19 July 1995, Cambridge.
Correspondence: Dr I. M. Anderson, University of Manchester Department of Psychiatry, Room 9809, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Tel: 0161 276 5396; Fax: 0161 273 2135; e-mail: ian.anderson{at}man.ac.uk
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ABSTRACT |
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Aims To investigate the effect of reducing brain serotonin function on anxiety at rest, and following 5% CO2 provocation in normal controls and patients with panic disorder.
Method Twenty drug-free patients with DSM III R panic disorder and 19 controls received a tryptophan-free amino acid drink on one occasion and a control drink on the other in a double-blind, balanced protocol. 5% CO2 was given as a panic challenge after 270 minutes.
Results Plasma tryptophan fell by more than 80% both patients and controls after the tryptophan-free drink. Tryptophan depletion did not alter resting anxiety. In patients alone, tryptophan depletion caused a greater anxiogenic response and an increased rate of panic attacks (9 v. 2, P<0.05) after 5% CO2 challenge. No normal volunteers panicked.
Conclusions Serotonin may directly modulate panic anxiety in patients with panic disorder. This may underlie the efficacy of serotonergic antidepressants in treating panic disorder.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Experimental procedures
Subjects were tested on two occasions separated by at least four days; for
females this occurred during the first 14 days of the menstrual cycle or in
the contraceptive pill-free week. On arrival, a forearm vein was cannulated
and kept patent using heparinised saline. After 45 minutes the subjects
received either a tryptophan-free 100 g amino acid drink or a control drink
containing 2.3 g of tryptophan at 09.00 (time zero). Each test session lasted
a further 330 minutes. A 5% CO2 panic challenge (see below) was
administered 270 minutes after the amino acid drink on both test days.
Psychological ratings and blood samples were taken before the amino acid drink
and then hourly until 240 minutes, followed by half-hourly until 330 minutes.
The subjects sat in a quiet testing room and were not allowed to sleep, eat or
smoke but could drink water and read emotionally neutral material. A meal was
provided at the end of the experiment.
Acute tryptophan depletion
Oral administration of a tryptophan-free amino acid load leads to a
profound reduction in plasma tryptophan concentration, thus reducing its
availability for brain 5-HT synthesis
(Reilly et al, 1997). In humans there is preliminary evidence for reduced 5-HT synthesis
(Nishizawa et al,
1997) and reduced cerebrospinal fluid tryptophan and
5-hydroxyindoleacetic acid (Carpenter
et al, 1998) after tryptophan depletion. Following a
standard protocol (Young et al,
1985; Benkelfat et al,
1994), subjects ate a low-protein diet (20 g) for 24 hours, fasted
overnight and received one of two amino acid drinks, double-blind, in a
balanced order in the morning. The drinks differed only in whether they lacked
or contained 2.3 g of tryptophan (tryptophan-free drink and control drink,
respectively). The other amino acids were: L-alanine, 5.5 g; L-arginine, 4.9
g; L-cysteine, 2.7 g; glycine, 3.2 g; L-histidine, 3.2 g; L-isoleucine, 8 g;
L-leucine, 13.5 g; L-lysine monohydrochloride, 11 g; L-methionine, 3 g;
L-phenylalanine, 5.7 g; L-proline, 12.2 g; L-serine, 6.9 g; L-threonine, 6.9
g; L-tyrosine, 6.9 g; and L-valine, 8.9 g. Methionine, cysteine and arginine
were encapsulated because of their unpleasant taste. Females received 80% of
the above amounts because of their lower body weight. The powdered amino acids
were mixed with 150 ml of water, 100 ml of chocolate syrup and two desert
spoons of sugar immediately before administration. Subjects drank the mixture
quickly through drinking straws and then chewed sugar-free gum to minimise the
unpleasant taste.
Panic challenge with 5% CO2
The CO2 challenge is a widely used panic challenge, with
evidence for increased sensitivity to its anxiogenic action in patients with
panic disorder (Sanderson & Wetzler,
1990) and those at genetic risk for panic disorder
(Perna et al, 1995).
This may reflect derangement of a brainstem suffocation-alarm system
(Klein, 1993). Using the
method of Roth et al
(1992), compressed air (21%
oxygen and 79% nitrogen from the British Oxygen Company) was administered for
10 minutes through a positive pressure mask before switching to continuous 5%
CO2 (5% CO2, 21% oxygen and 74% nitrogen; British Oxygen
Company) for 20 minutes. Single breaths of 5% CO2 were given at two
and seven minutes to maximise blindness. The gas cylinders were in an adjacent
room and connected to the subject through a Y-valve and
CO2-impervious tubing, with a reservoir bag connected to the tubing
just before the mask. End-tidal CO2 pressure was monitored. To
minimise psychological effects on panic rates
(Sanderson et al,
1989), the experimental conditions were standardised by
familiarising the subjects with the procedure on the first visit, providing
oral and written information on the effects of breathing 5% CO2 on
both test days and ensuring that the experimenter was visible during the
challenge. A prior decision was made to exclude subjects from analysis of the
effects of panic challenge if they panicked before CO2 was
administered.
Psychological measurements
Rating scales consisted of the Visual Analogue Scales (VAS: sad, anxious,
panicky, lightheaded, happy, drowsy, nauseated, irritable) on a 100 mm line (0
mm=not at all; 100 mm-extremely), the Spielberger State Anxiety Inventory
(STAIS; Spielberger et al,
1979) and Profile of Mood States (POMS;
McNair et al, 1971),
given in that order. Before and after the 5% CO2 challenge,
subjects completed a modified version of the Acute Panic Inventory (API;
Dillon et al, 1987),
consisting of 24 items formed from the 13 DSMIIIR panic symptoms
and apprehension rated on a five-point severity scale (0=not present;
4=extremely severe). After CO2 challenge, subjects were asked to
rate the peak severity that occurred during the challenge and also the
similarity to their usual panic attacks. Because of the difficulties in
assessing laboratory panic attacks
(Sanderson & Wetzler,
1990), we applied two definitions of panic attack. First, the
patients' own report of a panic attack rated at least quite
similar to their usual attacks. Second, an increase over baseline of
four DSMIIIR panic symptoms rated at least moderately severe on
the API, together with an increased Anxiety VAS or Panic VAS of 15 mm or
greater.
Biochemical measurements
Blood samples were taken into lithium heparin tubes and centrifuged within
an hour for 10 minutes at 2400 rpm and 4°C. The separated plasma was
frozen and stored at 20°C before analysis. Plasma was assayed for
total and free tryptophan (at 0, 240 and 300 minutes) and for cortisol (at
270, 300 and 330 minutes). Plasma tryptophan concentration was measured by a
semi-automated high-performance liquid chromatography with fluorescence
end-point detection. Intra- and interassay coefficients of variation were 8%
and 13%, respectively, and the limit of detection was 1.3 pg/ml. Cortisol was
analysed by standard radioimmunoassay. Intra- and inter-assay coefficients of
variation were 4.3% and 5.6%, and the limit of detection was 0.1 µg/100
ml.
Analysis
Two time periods were analysed using SPSS for Windows Release 6 (SPSS Inc.,
Chicago, IL): the pre-challenge period (0-270 minutes), reflected a resting
period; and the CO2 challenge period (270-330 minutes). The
principal analysis for continuous data was by repeated measured analysis of
variance (ANOVA) using the HuynhFeldt correction with a
between-subjects factor (group) and two within-subject factors of occasion
(tryptophan-free or control drink) and time. Controls and patients were
analysed separately by ANOVA following significant interactions by group or by
occasion. Post hoc t-tests were used to aid interpretation of data.
Categorical data were analysed using McNemar's test
(Armitage & Berry, 1987)
and correlations using Spearman's correlation coefficient. Data are presented
as mean (s.d.) values.
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RESULTS |
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Tryptophan measures
There were no significant differences in baseline measures
(Table 2). Significant occasion
x time interactions for total tryptophan (F(2,74)=174.23,
P<0.001) and free tryptophan (F(2,74)=4.123,
P<0.001) occurred, with plasma tryptophan decreasing on the
tryptophan depletion occasion (total tryptophan: patients, 83% and volunteers,
85%; free tryptophan: patients, 62% and volunteers, 74%) and increasing on the
control occasion (total tryptophan: patients, 283% and volunteers, 256%; free
tryptophan: patients, 164% and volunteers, 185%).
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Pre-challenge period (time 0-270 minutes)
Caseline behavioural ratings
Patients had significantly higher anxiety and depression ratings than
controls (group effect on ANOVA P<0.01 for all measures;
Fig. 1).
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Effect of tryptophan depletion on behavioural ratings
Anxiety scores, apart from Panic VAS, fell during the pre-challenge period
(Figs 1a-d). There were no
significant interactions, indicating no effect of tryptophan depletion and no
difference between patients and controls. Anxiety increased between 240 and
270 minutes (Fig. 1),
suggesting acute anticipatory anxiety before the CO2 challenge.
Analysis of variance of these two time points showed increases in STAIS
(time: F(1,37)=5.43; P=0.025) and POMS Anxiety-Tension
(time: F(1,37)=4.00; P=0.053). Patients, but not controls,
had increased Anxiety VAS ratings (group x time: F(1,37)=5.89;
P=0.02) with a similar but non-significant pattern with Panic VAS
(Fig. 1). For Panic VAS alone
there was a greater increase on the tryptophan depletion occasion compared
with the control occasion (occasion x time: F(1,37)=4.37;
P=0.043).
Depression-related ratings fell during this period but this was only significant for POMS Depression. There were no significant interactions between group, time or occasion. No significant effect of tryptophan depletion was found in separate analyses of patients with panic disorder according to the presence (n=7) or absence (n=13) of current major depression or a past history of major depression in the absence of current depression (n=8) (results not shown).
Other behavioural ratings generally showed a maximal effect at 60 minutes due to the drink, returning towards baseline by the end of the resting period. There were significant effects of time for Nausea VAS, Lightheaded VAS, Drowsiness VAS and POMS Vigour (all P<0.001) but not POMS Fatigue or POMS Confusion. There were no significant differences over time between patients and volunteers or occasions.
Carbon dioxide challenge period (270-330 minutes)
One patient discontinued the gas inhalation before receiving CO2
(control occasion) and one panicked at the time of putting on the mask
(tryptophan depletion occasion), leading to exclusion. One control subject was
excluded from analysis of API scores only because of a very high
pre-inhalation API score inconsistent with all other anxiety ratings.
Cortisol measures
Plasma samples from four controls were missing at 330 minutes and results
were therefore available from 32 subjects. There were no significant effects
of CO2 inhalation and no difference between patients and controls
or occasion. Patients who had a panic attack (by either definition) did not
differ from those not panicking (results not shown).
Psychological measures
As noted for other anxiety ratings (see above), patients had significantly
higher API scores at challenge baseline (270 minutes) than controls (see
Fig. 3; group:
F(1,34)=18.62; P<0.001). Carbon dioxide challenge caused
increases in all ratings of anxiety (Fig.
2). Significant group x time interactions occurred for all
anxiety ratings due to patients' ratings increasing more than controls
(Fig. 2). Panic VAS showed a
significant group x occasion x time interaction
(F(2,70)=5.69; P=0.005) due to a greater increase following
tryptophan depletion in patients but not controls. When controls and patients
were considered separately, the anxiogenic effect of CO2 was small
in controls and only Anxiety VAS increased significantly
(F(2,36)=4.36; P=0.024); indeed, STAIS decreased
throughout the period (F=(2,36)9.27; P=0.001). Tryptophan
depletion had no effect on anxiety ratings in controls. In contrast, patients
showed consistent increases in anxiety ratings following CO2
(Fig. 2), with greater
responses seen on the tryptophan depletion occasion for Panic VAS
(F(2,34)=6.14; P=0.005) and a trend for Anxiety VAS
(F(2,34)=3.07; P=0.060). Increases in POMS Anxiety-Tension
and STAIS were non-significantly greater on the depletion occasion.
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Carbon dioxide inhalation increased API scores particularly in patients and on the tryptophan depletion occasion (group x occasion x time: F(1,34)=7.90; P=0.008; Fig. 3). In controls analysed separately there was a modest CO2-induced increase in API scores (time: F (1,17)=6.54; P=0.021) but no effect of tryptophan depletion (occasion x time: F (1,17)=0.46; P=0.508). In patients there was a robust effect of CO2 inhalation (time: F(1,17)=46.08; P<0.001) and a marked effect of tryptophan depletion (occasion x time: F(1,34)=23.06; P<0.001).
Panic attacks in patients were more frequent on the tryptophan depletion occasion for both subjective panic attacks (eight v. two, P<0.05) and DSMIIIR panic attacks (nine v. two, P<0.05). Patients commented that the security of the experimental situation had reduced cognitive symptoms and severity compared with their usual panic attack. This is reflected in relative low mean panic attack similarity ratings, which were higher on the tryptophan depletion occasion (1.39 (s.d.=0.85) v. 0.78 (s.d.=0.65), P=0.012).
On the tryptophan depletion occasion, patients with a DSMIIIR panic attack had higher mean anxiety scores immediately before CO2 inhalation (270 minutes) than non-panickers; this was statistically significant for API scores (11.9 (s.d.=10.9) v. 3.8 (s.d.=2), P=0.049), STAIS (49.2 (s.d.=7.9) v. 38.4 (s.d.=9.2), P=0.017) and POMS Anxiety-Tension (12.1 (s.d.=5.0) v. 6.0 (s.d.=4.1), P=0.012). However, there were no significant differences at 240 minutes on any anxiety measures, suggesting that those who experienced greater acute anticipatory anxiety were more likely to panic when challenged by 5% CO2.
Analysis of the two respiratory items on the API separately showed no significant effect of tryptophan depletion (increases on control v. tryptophan depletion occasion: 2.0 (s.d.=1.7) v. 2.6 (s.d.=2.0), P=0.165 in patients and 0.8 (s.d.=1.5) v. 1.4 (s.d.=1.2), P=0.127 in volunteers).
Correlations
In patients, anxiety responses to 5% CO2 (the difference score
between 270 and 300 minutes) correlated with a number of values at baseline
(time zero) and 270 minutes on the tryptophan depletion occasion. Anxiety VAS
responses to 5% CO2 tended to correlate with: Anxiety VAS values at
time zero (rho=0.43, P=0.078) and 270 minutes (rho=0.44,
P=0.065); Panic VAS values at time zero (rho=0.57, P=0.050)
and 270 minutes (rho=0.51, P=0.031); and STAIS at time zero
(rho=0.56, P=0.016). Panic VAS responses also correlated with
STAIS at time zero (rho=0.49, P=0.039). No significant
correlations between baseline ratings and anxiety responses to 5%
CO2 challenge were seen on the control occasion or in
volunteers.
There were no consistent patterns of association between bichemical measures and anxiety responses to 5% CO2.
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DISCUSSION |
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Tryptophan depletion and resting anxiety and depression
We only measured resting anxiety up to 270 minutes, which could be too
short to detect an effect because maximum plasma tryptophan reduction does not
occur until about 300 minutes (Young
et al, 1985; Delgado
et al, 1990; see Table
2). However, our results agree with those of Goddard et
al (1994) who also found
little effect of tryptophan depletion in a small study with eight drug-free
patients with panic disorder assessed up to 420 minutes. They are also
consistent with findings in other psychiatric conditions where tryptophan
depletion does not alter anxiety (e.g.
Delgado et al, 1994;
Aronson et al, 1995;
Benkelfat et al,
1995), unless there is exacerbation of the primary disorder (e.g.
Delgado et al, 1990;
Menkes et al, 1994;
Weltzin et al, 1994).
The lack of effect of tryptophan depletion on anxiety in normal volunteers in
our study is also consistent with most other studies (e.g.
Young et al, 1985;
Smith et al, 1987; Weltzin et
al, 1994; Goddard et
al, 1995; Koszycki et
al, 1996).
Our findings appear to conflict with evidence suggesting that tryptophan depletion should be anxiolytic. Reducing 5-HT function is anxiolytic in a number of paradigms in animals (Coplan et al, 1992) and ritanserin, a 5-HT2 antagonist, is anxiolytic in a human model of generalised anxiety and in patients with mixed anxiety and depression (Deakin et al, 1992). Conversely, increasing 5-HT functioning using m-chlorophenylpiperazine (Charney et al, 1987) and fenfluramine (Targum & Marshall, 1989) challenge causes anxiety in patients with panic disorder, as can initial treatment with clomipramine and selective serotonin reuptake inhibitors (e.g. Ramos et al, 1993). These data are difficult to reconcile but the present study suggests that the increased levels of anxiety generally seen in patients with panic disorder are not simply due to tonically increased 5-HT function.
Our lack of effect of tryptophan depletion on depressive symptoms is consistent with the previous study in patients with panic disorder (Goddard et al, 1994), whereas in other patient groups there have been variable findings with regard to induction of depression (Delgado et al, 1990, 1994; Barr et al, 1994; Smith et al, 1997), possibly related to diagnosis and drug status. Similarly variable results in lowering mood have been seen in normal volunteers studies (e.g. Young et al, 1985; Oldman et al, 1994; Weltzin et al, 1994; Goddard et al, 1995; Koszycki et al, 1996).
Tryptophan depletion and provoked anxiety
We found 5% CO2 inhalation to be panicogenic in patients but
only mildly anxiogenic in controls. Like Roth et al
(1992), we found that patients
who panicked had higher anxiety levels immediately before challenge than
non-panickers. They argued that this could be explained by anticipatory
anxiety increasing above a threshold of tolerance (i.e. situational panic). In
contrast, other studies have found no relationship between baseline anxiety
and subsequent panic attacks (e.g. Gorman
et al, 1988;
Sanderson & Wetzler,
1990), suggesting a model of spontaneous panic attacks. To what
extent prior anxiety may predict panic to CO2 challenge therefore
remains unresolved, but our results suggest that it may act as a factor.
Tryptophan depletion increased acute anticipatory and 5% CO2-induced anxiety in patients and it is interesting to relate this to the effect of treatment with 5-HT-enhancing antidepressants, which have been shown to reduce panic patients' hypersensitivity to CO2 challenge (Bertani et al, 1997; Gorman et al, 1997). Our result supports increased 5-HT neurotransmission playing an important part in the anti-panic effect of these drugs. The lack of effect that we found in volunteers appears to contrast with a recent report of increased anxiety ratings and some panic symptoms following 35% CO2 inhalation after tryptophan depletion (Klaassen et al, 1998). Although methodological differences, including the weak anxiogenic response in our study, may explain this, there have been conflicting results in normal volunteers using other anxiety challenges that do not simply appear related to the severity of the anxiogenic challenge. Tryptophan depletion has been reported to enhance anxiety after yohimbine administration (Goddard et al, 1995) but not after cholecystokinin challenge (Koszycki et al, 1996) or in response to simulated public speaking (Mortimore et al, 1997). It therefore remains unclear whether 5-HT modulates stimulated anxiety in normal subjects.
It is of interest that the measures specifically relating to panic (Panic VAS and API) appeared to be more robustly affected than general anxiety ratings in patients. We cannot, however, distinguish between situational panic related to anticipation of a proximal threat (imminent or actual CO2 challenge) and panic produced by a biological effect of CO2. Although the results are generally consistent with the hypothesis that 5-HT acts to restrain panic, a distinction between its role in spontaneous panic compared to acute anticipatory anxiety is difficult to sustain and it may be that the important distinction is between other aspects of anxiety, such as proximal versus distal threat or conditioned versus unconditioned anxiety.
The lack of effect of 5% CO2 challenge on plasma cortisol is generally consistent with the literature (Carr et al, 1986; Klein, 1993). Why patients with panic disorder lack a stress hormone response when panicking is unknown, but it could relate to an adaptive change in the hypothalamus-pituitary axis related to chronic anxiety or stress (Gray, 1987), or simply a low threshold for reporting panic attacks. In addition, the anxiety responses to 5% CO2 were relatively mild and may have been insufficient to stimulate a stress response.
Methodological considerations
We found a lower rate of panic following 5% CO2 inhalation on
the control occasion than that suggested by the literature (about 10%
v. 50%; Sanderson & Wetzler,
1990), possibly because the setting of the experiment and careful
explanation minimised the panic rate through cognitive factors
(Clark, 1986). We think it
unlikely that the control drink suppressed the panic rate, because although it
increased both total and free plasma tryptophan, the plasma tryptophan to
large neutral amino acid ratio and hence tryptophan entry into the brain would
still be expected to be reduced, although much less than following the
tryptophan-free drink (Weltzin et
al, 1994).
The successful blinding of the experimental occasions argues against cognitive factors directly affecting the result. However, there is a theoretical possibility that tryptophan depletion stimulated respiratory function and hence, indirectly, the panic rate through the occurrence of somatic symptoms. In rats, 5-HT depletion stimulates respiration (Olson et al, 1979) and a similar tendency was seen with tryptophan depletion in humans (Kent et al, 1996). However, arguing for a direct effect on anxiety, tryptophan depletion enhanced acute anticipatory anxiety and there was no significant effect on the respiratory item scores of the API. Unfortunately we did not have direct measures of respiratory effort or tidal volume for a direct answer to the question.
Implications
Our study suggests that 5-HT directly inhibits panic anxiety and that this
may help to explain the anti-panic effects of antidepressants such as
selective serotonin reuptake inhibitors. The lack of effect of tryptophan
depletion on resting anxiety is consistent with 5-HT playing a different role
in different types of anxiety, although the results did not suggest a direct
role in the maintenance of high resting levels of anxiety in our patients with
panic disorder. This implies that the anxiolytic effect of antidepressants in
generalised anxiety and non-panic anxiety associated with depression may
involve a different mechanism to the anti-panic effect. Studies using
different manipulations of 5-HT function and alternative anxiety challenges in
patients with anxiety disorders and healthy volunteers are needed in order to
shed further light on the role of 5-HT in human anxiety.
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Clinical Implications and Limitations |
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LIMITATIONS
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Received for publication June 18, 1999. Revision received September 9, 1999. Accepted for publication September 10, 1999.