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Evening salivary alpha-amylase, major depressive disorder, and antidepressant use in the Netherlands Study of Depression and Anxiety (NESDA)
Psychiatry Research 208 (2013) 41–46
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Evening salivary alpha-amylase, major depressive disorder, and antidepressant use in the Netherlands Study of Depression and Anxiety (NESDA) Gerthe Veen a,b,n, Erik J. Giltay b, Carmilla M.M. Licht a, Sophie A. Vreeburg a, Christa M. Cobbaert c, Brenda W.J.H. Penninx a,b,d, Frans G. Zitman b a
Department of Psychiatry, VU University Medical Center, Amsterdam, The Netherlands Department of Psychiatry, Leiden University Medical Center, Leiden, The Netherlands Department of Clinical Chemistry, Leiden University Medical Center, Leiden, The Netherlands d Department of Psychiatry, University Medical Center Groningen, Groningen, The Netherlands b c
art ic l e i nf o
a b s t r a c t
Article history: Received 28 January 2012 Received in revised form 11 March 2013 Accepted 17 March 2013
Salivary alpha-amylase (sAA) may be a suitable index for sympathetic activity and dysregulation of the autonomic nervous system. The relationship between antidepressants and depression with sAA levels was studied, since antidepressants were previously shown to have a profound impact on heart rate variability as an ANS indicator. Data are from 1692 participants of the Netherlands Study of Depression and Anxiety (NESDA) who were recruited from the community, general practice, and specialized mental health care. Differences in evening sAA levels were examined between patient groups (i.e., 752 current major depressive disorder [MDD], 611 remitted MDD, and 329 healthy controls) and between 46 tricyclic antidepressant (TCA) users, 307 selective serotonin reuptake inhibitor (SSRI) users, 97 users of another antidepressant, and 1242 non-users. Each participant sampled twice at 22.00h and 23.00h. In multivariable analysis, there was a trend over the three groups with increasing sAA levels from controls to remitted MDD to current MDD that approached significance. Furthermore, in comparison to non-users of antidepressants, TCA rather than SSRI users showed higher sAA levels, that persisted after multivariable adjustment. The present study shows that higher evening sAA levels in depressed patients, indicative of an increased sympathetic activity, may be induced by TCAs. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Autonomic nervous system Stress Catecholamines Heart rate Heart rate variability
1. Introduction There is a central belief that a dysregulated autonomic nervous system (ANS) plays a role in the pathophysiology of depression. In previous cross-sectional studies, diverse methods of measuring ANS tone and reactivity have been used, including plasma levels of catecholamines (i.e. (nor)epinephrine), heart rate, and heart rate variability (HRV). Recently, salivary alpha-amylase (sAA) assessments have been proposed as another noninvasive technique for the measurement of the sympathetic tone of ANS (Chatterton Jr. et al., 1996; Granger et al., 2007). Summarizing the existing literature, early studies suggested sAA as a noninvasive marker of adrenergic activity in humans (Chatterton et al., 1996; Skosnik et al., 2000; Nater and Rohleder, 2009). Activation of the
n Corresponding author at: Department of Psychiatry, VU University Medical Center, Amsterdam, The Netherlands. Tel.: þ 31 20 7884666; fax: þ 31 20 7884677. E-mail address: [email protected] (G. Veen).
0165-1781/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.psychres.2013.03.012
autonomic nervous system results in norepinephrine release that may subsequently elicit the release of alpha-amylase by the salivary glands. The underlying function of the norepinephrine is to balance vigilance/scanning behavior with focused attention on novel environmental stimuli and the state of arousal. Norepinephrine is involved intrinsically with the stress response system, and high levels of norepinephrine have been implicated in the pathogenesis of depressive disorders (Goddard et al., 2010). Under basal conditions in healthy volunteers, sAA activity shows a diurnal profile with a decrease during the first 30 min after awakening and an increase during the rest of the day (Nater et al., 2007; Rohleder et al., 2004a, 2004b). Diurnal sAA levels were relatively independent of several possible confounders like age, gender, body mass index (BMI), activity level, smoking, eating and drinking but significantly associated with chronic stress and stress reactivity in healthy volunteers (Nater et al., 2007). With regard to evening sAA levels, only age and alcohol intake were found to be significant determinants (Veen et al., 2012). Different types of psychological and physiological challenges were followed by
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G. Veen et al. / Psychiatry Research 208 (2013) 41–46
increases in sAA (Chatterton Jr. et al., 1996, 1997; Kivlighan and Granger, 2006; Nater et al., 2005; Nater and Rohleder, 2009). sAA might be a better alternative to assess ANS than plasma catecholamines, because catecholamines are sampled from the antecubital venous blood, which is often perceived as stressful and may rather reflect local sympathetic activity in the forearm rather than total body sympathetic activity (Veith et al., 1984). This may explain the poor correlation of plasma catecholamines and sAA (Nater et al., 2006). However, significant associations were found between cardiac measures of ANS, i.e. heart rate and heart rate variability (HRV) and sAA (Bosch et al., 2003; Nater et al., 2006), yielding sAA as an interesting proxy of ANS, although sAA in only partial under B-adrenergic control. sAA may be an interesting alternative to cardiac measures, because it can be sampled rather easily and stress-free at home. Previous studies on ANS function in depression showed a dysfunction of the autonomic system through higher levels of catecholamines (Veith et al., 1994), and lower heart rate variability (Licht et al., 2008; Veith et al., 1994) although the latter was for a large part due to antidepressant effects. As far as we know, one study on sAA was performed in current depression (Tanaka et al., 2012) and one study in remitted depression (Bagley et al., 2011). They found that in 47 female patients with a current depression sAA levels were significantly elevated relative to controls both before and after electrical stimulation. In 22 patients with remitted depression no difference in sAA reactivity on stress was found compared to controls. In both studies sAA was measured in response to a stress test, while our analysis focused on basal sAA levels. With regard to anxiety and eating disorders, hyperactivity of ANS, as measured with sAA, was found in studies on generalized social anxiety disorder and anorexia nervosa (van Veen et al., 2008; Monteleone et al., 2011). In the present study, we investigated whether there is a difference in basal ANS in patients with a major depressive disorder (MDD) compared to healthy controls. We hypothesized that the ANS, as measured using sAA, would show hyperactivity in depression, leading to higher sAA levels. Furthermore, we studied effects of antidepressants on sAA levels, since we earlier found that antidepressants had an extensive impact on ANS indicators (Licht et al., 2008; Veith et al., 1994). In this paper, we report on sAA acquired at two time points in the late evening (i.e., at 22:00h and 23:00h) to derive measures of the mean evening sAA levels. The use of late evening sAA levels yields the opportunities to study sAA at its peak values of the diurnal secretion pattern (Nater and Rohleder, 2009). This decreases the effects of measurement error and the episodic nature of sAA release, and reduces the potential influence of confounding by carbohydrate consumption and physical activity (Rohleder and Nater, 2009). In this article, we report on the associations between use of antidepressants, major depressive disorder and evening sAA.
major depressive disorder (MDD), and persons with current MDD. Control participants were defined as having no lifetime history of depressive disorder (MDD or dysthymia) or anxiety disorder (panic disorder, generalized anxiety disorder, agoraphobia, or social phobia) as assessed by the DSM-IV Composite International Diagnostic Interview (CIDI) version 2.1, no family history of depression, and a score below 14 on the 30-item Inventory of Depressive Symptomatology, Self-Report (IDS-SR) (Rush et al., 1996). These criteria were fulfilled by 413 respondents from the Netherlands Study of Depression and Anxiety respondents. Persons with remitted MDD (n ¼810) had a history of MDD but no diagnosis of MDD in the past 6 months as diagnosed by the CIDI. The third MDD group consisted of 1115 participants with a current diagnosis of MDD, i.e., in the past 6 months, as assessed by the CIDI (of whom 802 [71.9%] had been diagnosed with MDD in the past month). Of these participants, 1692 (72.4%) (329 control participants, 611 participants with remitted MDD, and 752 participants with current MDD) returned saliva samples (see below). Respondents on saliva collection did not differ from non-respondents in gender, but were older (age, 44.1 vs 38.2 years, respectively; Po 0.001), had a higher level of education (12.4 vs 11.6 years of education, respectively; Po 0.001) and less likely to be currently depressed (44.2% vs 57.4%, respectively; Po 0.001). Prior to participation, written informed consent was obtained from all participants. The study was approved by the ethics committee of the Leiden University Medical Center.
2.2. Salivary alpha-amylase At the baseline interview participants were instructed to collect saliva samples at home on a regular (preferably working) day shortly after the interview. The median time between the interview and saliva sampling was 9 days (25th–75th percentile, 4–22 days). Instructions prohibited eating, smoking, drinking, or brushing teeth within 15 min. Saliva samples were obtained using Salivettes (Sarstedt AG and Co, Nümbrecht, Germany) at two time points. Two evening samples were collected at 22:00 h and 23:00 h. Samples were stored in refrigerators and returned by mail. After receipt, salivettes were centrifuged, aliquoted, and stored at −80 1C. For assaying, after overnight thawing of the saliva at 4 1C, samples were diluted 50-fold with a Hamilton Microlab 500 B/C dilutor in physiological saline solution (Versylenes Fresenius, Cat. Nr. B230551). Analyses were performed using a kinetic colorimetric assay for total amylase activity (Cat Nr. 03183742, Roche Diagnostics, Mannheim, Germany) on a routine clinical chemistry analyzer. The total amylase assay is standardized to the IFCC reference measurement procedure (Lorentz, 1998), guaranteeing worldwide comparability of the data. Amylase activities are measured and expressed in IU/L at 37 1C. Within-run imprecision for the control pool ranged from 0.7% to 2.7% for the combined predilution step and the analysis across the entire study period. Between-run analytical imprecision was lower than 5% throughout this study.
2.3. Covariates We previously described significant determinants of sAA evening levels, i.e. age and alcohol intake (Veen et al., 2012). These identified determinants were considered as covariates in the present study. Alcohol consumption was expressed as number of units of alcohol per day. In addition we added gender as covariate.
2.4. Antidepressant use Medication use at the baseline assessment was collected via subject self-report (7 19%) and medication containers ( 7 81%) brought to the interview. Using the Anatomical Therapeutic Chemica (ATC) classification (www.whocc.no/atcddd/), psychoactive medication was categorized into antidepressants (tricyclic antidepressants [N06AA], selective serotonin reuptake inhibitors [N06AB], and other antidepressants [N06AF, N06AG, N06AX]), and benzodiazepines (N03AE, N05BA, N05CD, N05CF).
2. Method 2.5. Statistical analysis 2.1. Participants Data are from the Netherlands Study of Depression and Anxiety, a large cohort study on the course of depressive and anxiety disorders. In total, 2981 respondents were recruited from the community, general practice care, and specialized mental health care. The study sample included persons with psychopathological findings as well as control participants without a psychiatric diagnosis. For objectives and methods of the Netherlands Study of Depression and Anxiety, see the article by Penninx et al. (2008). The research protocol was approved by the ethical committee of participating universities, and all of the respondents provided written informed consent. To investigate whether sAA levels were different in depressed versus nondepressed respondents, taking into account remission of symptoms, three comparison groups (MDD status) were formed: control participants, persons with remitted
The distribution of sAA values was strongly positively skewed and therefore log-transformed values were used for analyses. Back-transformed geometric mean values are presented in tables and figures. On average, participants who provided both samples (n¼ 1659) showed higher sAA levels at 22:00 h compared to 23:00 h (t(1658) ¼12.0, po 0.001, r¼ 0.78). Because of the high correlation, the mean value of sAA levels at 22:00 h and 23:00 h was used in further analyses, to reduce the variability. When only one sample was provided (19 participants provided only a 22.00 h sample and 14 participants only a 23.00 h sample), that value was used. Baseline characteristics of the three MDD groups were compared using χ2 tests for categorical variables and one-way ANOVA for continuous variables. Multivariable analyses of covariance (ANCOVA) were used to examine differences in sAA levels across groups. All of the analyses were conducted using SPSS version 17.0 statistical software (SPSS Inc, Chicago, Illinois).
G. Veen et al. / Psychiatry Research 208 (2013) 41–46
3. Results 3.1. Clinical characteristics Participants were on average 44.3712.3 years old (range 18–65), and 66.5% were women. Characteristics across groups are presented in Table 1. Participants with remitted (n¼611) or current MDD (n¼752) were more often female and younger than controls (n¼ 329). Of the participants with current MDD, 210 (28.1%) were severely depressed as defined by an IDS score of 39 or higher, corresponding to a score of 20 or higher on the 17 item Hamilton Depression Scale. Of the participants with current MDD, 439 used no antidepressant, 28 were tricyclic antidepressant (TCA) users, 212 were selective serotonin reuptake inhibitor (SSRI) users and 73 used another antidepressant (3 duloxetine, 51 venlafaxine, 17 mirtazapine, 1 trazodone, 1 tranylcypromine). Of the participants with remitted MDD, 474 used no antidepressant, 18 were TCA users, 95 were SSRI users and 24 used another antidepressant (20 venlafaxine, 4 mirtazapine). Only one patient was treated with a non-selective monoamino-oxydase inhibitor (MAOI; tranylcypromine), and therefore its effects on sAA could not be ascertained in our study.
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persisted (p ¼0.06) (Table 2 and Fig. 1). When repeating the analyses in non-users of antidepressants no significant differences were found (Table 2). When using a continuous measure of depression severity (IDS) no association with sAA was found (r ¼0.02, p¼ 0.36). 3.3. Antidepressant use and salivary alpha-amylase In comparison to participants without antidepressants (221 U/ l; 95% CI: 211–232), users of TCAs showed significantly higher sAA levels (348 U/l; 95% CI: 271–447, p o0.001). After adjustment for age, gender, and alcohol intake these differences remained significant (p ¼0.003), even after additional adjustment for group (controls, remitted MDD, current MDD) (p ¼0.01) (Table 3 and Fig. 1). Users of SSRIs and other ADs did not show changes in sAA levels compared to participants without antidepressants (respectively, p ¼0.12 and p ¼0.27). When comparing the 74 users of serotonin-norepinephrine reuptake inhibitors (SNRIs: subgroup of the other AD users) to the 1242 participants not using antidepressants in a sensitivity analysis, the sAA levels were not statistically significantly higher in the SNRI group in the adjusted model 2 (mean 263; 95% CI: 216–312 vs mean 224 U/L; 95% CI: 213–235: p¼ 0.12).
3.2. MDD status and salivary alpha-amylase In unadjusted analyses, the geometric mean sAA was slightly higher in current MDD (230 U/l; 95% confidence interval [CI]: 216– 245) compared to remitted MDD (221 U/l; 95% CI: 206–237) and controls (214 U/l; 95% CI: 195–235), but these differences in sAA levels did not reach significance. However, a trend toward significance (p ¼0.06) was found for sAA when comparing participants with current MDD with controls after adjustment for age, gender, and daily alcohol intake. After additional adjustment for antidepressant use, this trend that approached significance
4. Discussion The present study showed that users of TCAs had significantly higher sAA levels compared with participants not using antidepressants, which remained significant after adjustment for covariates, i.e., gender, age, alcohol intake and group (controls, remitted MDD, and current MDD). We are not aware of previous studies that investigated this association. Additionally, a trend toward significance was found with higher sAA levels in participants
Table 1 Characteristics according to diagnoses of depressive disorders in 1692 participants. Demographic variables
Controls
Remitted MDD
Current MDD
P
No. of participants Female (%) Age (years, mean, S.D.) Daily alcohol intake (in units) (mean, S.D.) Antidepressant use TCA (%) SSRI (%) Other (%) Benzodiazepine use (%)
329 59.3 47.6 (11.7) 1.1 (1.3)
611 70.4 44.9 (12.5) 1.0 (1.4)
752 60.5 42.2 (12.1) 0.9 (1.4)
0.003 o 0.001 0.08
– – – 2.4
2.9 16.2 4.3 12.4
3.7 28.2 10.5 23.0
0.02 o 0.001 o 0.001 o 0.001
Data are presented as means 7 standard deviation (S.D.) or percentage. P-values of one-way ANOVAs and Chi-Square tests are presented. Abbreviations: MDD¼ major depressive disorders; TCA ¼ tricyclic antidepressant; SSRI ¼ selective serotonin reuptake inhibitor.
Table 2 Salivary alpha-amylase levels in controls, remitted and current MDD patients.
All participants No. Unadjusted mean (U/ml) Adjusted mean (Model 1) (U/ml) Adjusted mean (Model 2) (U/ml) Only non-users of antidepressants No. Unadjusted mean (U/ml) Adjusted mean (Model 1) (U/ml)
Controls
Remitted MDD
Pa
Current MDD
Pa
329 214 (195–235) 209 (190–230) 208 (189–230)
611 221 (206–237) 220 (206–236) 220 (206–236)
0.59 0.38 0.36
752 230 (216–245) 233 (219–248) 234 (219–249)
0.20 0.06 0.06
329 214 (195–235) 209 (190–230)
474 234 (207–242) 224 (207–241)
0.46 0.24
439 223 (206–242) 228 (211–248)
0.50 0.13
Data are presented as back-transformed geometric estimated means with 95% confidence intervals within groups. Pa: simple contrast p-value of patient group vs controls are presented. Model 1: adjusted for age, gender, and alcohol intake. Model 2: Additionally adjusted for kind of antidepressants (no antidepressants, TCA, SSRI, and other antidepressants).
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G. Veen et al. / Psychiatry Research 208 (2013) 41–46
(Tulen et al., 1996) with current depression compared to control participants, but only after adjustment for covariates, i.e., gender, age and alcohol intake. Our findings consistent with those from Licht et al. (2008), who found that TCA use was strongly associated with ANS measures, e. g. heart rate variability, heart rate, blood pressure, and pre-ejection period. Some supporting findings were also found in animal data. In four studies, effects of TCAs were studied on salivary glands and oral microbiota in rats. These studies showed that there was a stimulating effect on the biosynthesis and secretion of alphaamylase (Koller et al., 2000,2001;Yu, 1992;Yu et al., 1989). This is in line with our findings. In another study the effects of salivary components of the tricyclic antidepressant imipramine (among other antidepressants) were studied. In this study sub-acute and chronic treatments of rats with the tricyclic antidepressant imipramine did not affect total protein and amylase levels (Kopittke et al., 2005. We did not find significant associations with the use of SSRIs and other antidepressants. In addition, other studies have also shown that the sympathetic part of ANS activity is higher in subjects using TCA than non-users (Barton et al., 2007; Lederbogen et al., 2001; Licht et al., 2010; van Zyl et al., 2008; Veith et al., 1994). The putative enhancing effects of TCAs on
Evening Salivary Alpha-Amylase (U/L)
400 380 360 340 320 300
P=0.06
280 260
P=0.36
240 220 200
P=0.01 P=0.12
0
=9 7
TC
A
A o
Cu r
N
us n= e SS 46 R Iu s n= e O th 30 7 er AD us n e
n= D 1. 24 2
52
=7
D n= D 61 1 re nt M D D n
9
M ed
em itt R
C
32
on t ro n= ls
P=0.27
Fig. 1. Salivary alpha-amylase across groups and kind of antidepressants on a logarithmic scale. The size of each square is proportional to the number of participants. Vertical lines indicate standard errors. P-values of multivariable analyses of covariance (ANCOVA) are presented after adjustment for age, gender, alcohol intake, and antidepressants use or group. Abbreviations: MDD ¼major depressive disorder; AD¼ antidepressants; TCA ¼ tricyclic antidepressants; SSRI¼ selective serotonin reuptake inhibitor.
sympathetic activity may have confounded the reported relationships between ANS and depression and anxiety in previous studies (Carney et al., 2005; Martens et al., 2008), which found elevated levels of plasma and urinary catecholamines, higher heart rate, and lower heart rate variability. A possible explanation for the effect of TCAs on ANS is that TCAs mainly inhibit the reuptake of norepinephrine, which increases its levels not only in the brain but also in the systemic circulation. Through accentuated antagonism norepinephrine has a blocking effect on muscarinic receptors, decreasing parasympathetic activity. In addition, the intrinsic anticholinergic effects of TCAs may even further inhibit parasympathetic activity (Esler et al., 1985; Manabe et al., 1991). In conclusion, lowered parasympathetic activity and increased sympathetic activity might be an effect induced by TCAs. We did not find convincing associations between sAA and depression, but only an association of borderline significance between participants with current MDD and controls. And when comparing MDD nonusers of antidepressants and controls no significant differences in sAA levels were found. Previous studies have shown mixed results. In studies that assessed sympathetic activity in depression employing non-cardiac specific measures such as norepinephrine spillover and skin conductance levels have yielded variable results. Most of these studies reported moderate increases in sympathetic activity in depression (Esler et al., 1985; Koschke et al., 2009; Veith et al., 1994), although some studies did not reveal any relationship (Barton et al., 2007; Guinjoan et al., 1995). Several studies suggest that beta-adrenergic activity or number of beta-receptors is decreased in depression (Bruno et al., 1983; Girdler et al., 1993; Krittayaphong et al., 1996; Light et al., 2009). This means that an increased sympathetic drive may be hidden by a decrease of effectiveness of the target organs to respond to the adrenergic drive. Some limitations should be recognized. First, sAA was assessed only in the evening, and therefore no statements can be made about associations between morning sAA and depression. Second, this study precludes drawing conclusions about whether sAA alterations in depression are more pronounced in total output (for which evening samples might be good proxy) or in change in daily rhythm. Multiple days of sampling may be necessary to reliably measure biological markers of the stress system (Hellhammer et al., 2007), but this was not an option in our large-scale study. Third, saliva for the assessment of sAA was collected using salivettes and participants were instructed to chew on the cotton roll, while active chewing rather than a passive drool may alter sAA (DeCaro, 2008). However, stress-induced increase of sAA levels has been shown to be independent of salivary flow rate (Rohleder et al., 2006). Fourth, because these analyses were crosssectional, our results do not indicate any causal directions of the associations found. Therefore, the higher sAA levels might be a consequence of depression or the treatment of depression. Our findings with respect to antidepressants might point in this
Table 3 Salivary alpha-amylase levels in non-users of AD, and in TCA, SSRI, and other AD users.
No. of participants Unadjusted mean (U/l) Adjusted mean (U/l) (Model 1) Adjusted mean (U/l) (Model 2)
No AD
TCA
Pa
SSRI
Pb
Other AD
Pc
1242 221 (211–232) 222 (211–233) 224 (213–235)
40 348 (271–447) 326 (254–418) 319 (248–409)
o 0.001 0.003 0.01
307 210 (191–231) 210 (190–231) 205 (185–226)
0.36 0.30 0.12
97 258 (217–307) 255 (215–302) 248 (208–295)
0.09 0.13 0.27
Data are presented as back-transformed geometric estimated means with 95% confidence intervals within groups. Pa: simple contrast p-value of TCA users vs non antidepressant users are presented. Pb: simple contrast p-value of SSRI users vs non antidepressant users are presented. Pc: simple contrast p-value of other AD users vs non antidepressant users are presented. Model 1: adjusted for age, gender, and alcohol intake. Model 2: additionally adjusted for group (controls, remitted MDD, current MDD). Abbreviations: MDD¼ major depressive disorders; TCA ¼ tricyclic antidepressant; SSRI ¼ selective serotonin reuptake inhibitor; AD ¼ antidepressant.
G. Veen et al. / Psychiatry Research 208 (2013) 41–46
direction. Fifth, there is still some controversy whether sAA is a valid and reliable measure of sympathetic activity of the ANS. Although there is growing evidence (Nater et al., 2005; Rohleder et al., 2006; Thoma et al., 2012), some issues remain unanswered, such as the relationship between salivary flow rate and parasympathetic activation, and other methodological aspects (see comments by Bosch et al. (2011)). Sixth, we did not have information on how long non-treated MDD patients did not take antidepressants and whether the two groups of patients (remitted vs current MD) differed in this variable. Lastly, although we assessed that depressed participants in our study had generally higher levels of stress and mood symptoms – as illustrated by higher IDS scores – compared with control participants, day-to-day variation that we could not further address could play an additional role in ANS function. Despite these limitations, our study had many strong aspects, including the large sample size with clearly distinct depression groups, the use of a recently introduced new measurement of ANS function, namely sAA, and the adjustment of various covariates. We conclude that higher sAA levels in depressed patients, indicative of an increased sympathetic activity, may be induced by TCAs. In addition, prospective studies are needed because of the high between-person variability. By using prospective study designs, within-patient comparisons over time of sAA levels would substantially increase the statistical power.
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Evening salivary alpha-amylase, major depressive disorder, and antidepressant use in the Netherlands Study of Depression and Anxiety (NESDA) Gerthe Veen a,b,n, Erik J. Giltay b, Carmilla M.M. Licht a, Sophie A. Vreeburg a, Christa M. Cobbaert c, Brenda W.J.H. Penninx a,b,d, Frans G. Zitman b a
Department of Psychiatry, VU University Medical Center, Amsterdam, The Netherlands Department of Psychiatry, Leiden University Medical Center, Leiden, The Netherlands Department of Clinical Chemistry, Leiden University Medical Center, Leiden, The Netherlands d Department of Psychiatry, University Medical Center Groningen, Groningen, The Netherlands b c
art ic l e i nf o
a b s t r a c t
Article history: Received 28 January 2012 Received in revised form 11 March 2013 Accepted 17 March 2013
Salivary alpha-amylase (sAA) may be a suitable index for sympathetic activity and dysregulation of the autonomic nervous system. The relationship between antidepressants and depression with sAA levels was studied, since antidepressants were previously shown to have a profound impact on heart rate variability as an ANS indicator. Data are from 1692 participants of the Netherlands Study of Depression and Anxiety (NESDA) who were recruited from the community, general practice, and specialized mental health care. Differences in evening sAA levels were examined between patient groups (i.e., 752 current major depressive disorder [MDD], 611 remitted MDD, and 329 healthy controls) and between 46 tricyclic antidepressant (TCA) users, 307 selective serotonin reuptake inhibitor (SSRI) users, 97 users of another antidepressant, and 1242 non-users. Each participant sampled twice at 22.00h and 23.00h. In multivariable analysis, there was a trend over the three groups with increasing sAA levels from controls to remitted MDD to current MDD that approached significance. Furthermore, in comparison to non-users of antidepressants, TCA rather than SSRI users showed higher sAA levels, that persisted after multivariable adjustment. The present study shows that higher evening sAA levels in depressed patients, indicative of an increased sympathetic activity, may be induced by TCAs. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Autonomic nervous system Stress Catecholamines Heart rate Heart rate variability
1. Introduction There is a central belief that a dysregulated autonomic nervous system (ANS) plays a role in the pathophysiology of depression. In previous cross-sectional studies, diverse methods of measuring ANS tone and reactivity have been used, including plasma levels of catecholamines (i.e. (nor)epinephrine), heart rate, and heart rate variability (HRV). Recently, salivary alpha-amylase (sAA) assessments have been proposed as another noninvasive technique for the measurement of the sympathetic tone of ANS (Chatterton Jr. et al., 1996; Granger et al., 2007). Summarizing the existing literature, early studies suggested sAA as a noninvasive marker of adrenergic activity in humans (Chatterton et al., 1996; Skosnik et al., 2000; Nater and Rohleder, 2009). Activation of the
n Corresponding author at: Department of Psychiatry, VU University Medical Center, Amsterdam, The Netherlands. Tel.: þ 31 20 7884666; fax: þ 31 20 7884677. E-mail address: [email protected] (G. Veen).
0165-1781/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.psychres.2013.03.012
autonomic nervous system results in norepinephrine release that may subsequently elicit the release of alpha-amylase by the salivary glands. The underlying function of the norepinephrine is to balance vigilance/scanning behavior with focused attention on novel environmental stimuli and the state of arousal. Norepinephrine is involved intrinsically with the stress response system, and high levels of norepinephrine have been implicated in the pathogenesis of depressive disorders (Goddard et al., 2010). Under basal conditions in healthy volunteers, sAA activity shows a diurnal profile with a decrease during the first 30 min after awakening and an increase during the rest of the day (Nater et al., 2007; Rohleder et al., 2004a, 2004b). Diurnal sAA levels were relatively independent of several possible confounders like age, gender, body mass index (BMI), activity level, smoking, eating and drinking but significantly associated with chronic stress and stress reactivity in healthy volunteers (Nater et al., 2007). With regard to evening sAA levels, only age and alcohol intake were found to be significant determinants (Veen et al., 2012). Different types of psychological and physiological challenges were followed by
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G. Veen et al. / Psychiatry Research 208 (2013) 41–46
increases in sAA (Chatterton Jr. et al., 1996, 1997; Kivlighan and Granger, 2006; Nater et al., 2005; Nater and Rohleder, 2009). sAA might be a better alternative to assess ANS than plasma catecholamines, because catecholamines are sampled from the antecubital venous blood, which is often perceived as stressful and may rather reflect local sympathetic activity in the forearm rather than total body sympathetic activity (Veith et al., 1984). This may explain the poor correlation of plasma catecholamines and sAA (Nater et al., 2006). However, significant associations were found between cardiac measures of ANS, i.e. heart rate and heart rate variability (HRV) and sAA (Bosch et al., 2003; Nater et al., 2006), yielding sAA as an interesting proxy of ANS, although sAA in only partial under B-adrenergic control. sAA may be an interesting alternative to cardiac measures, because it can be sampled rather easily and stress-free at home. Previous studies on ANS function in depression showed a dysfunction of the autonomic system through higher levels of catecholamines (Veith et al., 1994), and lower heart rate variability (Licht et al., 2008; Veith et al., 1994) although the latter was for a large part due to antidepressant effects. As far as we know, one study on sAA was performed in current depression (Tanaka et al., 2012) and one study in remitted depression (Bagley et al., 2011). They found that in 47 female patients with a current depression sAA levels were significantly elevated relative to controls both before and after electrical stimulation. In 22 patients with remitted depression no difference in sAA reactivity on stress was found compared to controls. In both studies sAA was measured in response to a stress test, while our analysis focused on basal sAA levels. With regard to anxiety and eating disorders, hyperactivity of ANS, as measured with sAA, was found in studies on generalized social anxiety disorder and anorexia nervosa (van Veen et al., 2008; Monteleone et al., 2011). In the present study, we investigated whether there is a difference in basal ANS in patients with a major depressive disorder (MDD) compared to healthy controls. We hypothesized that the ANS, as measured using sAA, would show hyperactivity in depression, leading to higher sAA levels. Furthermore, we studied effects of antidepressants on sAA levels, since we earlier found that antidepressants had an extensive impact on ANS indicators (Licht et al., 2008; Veith et al., 1994). In this paper, we report on sAA acquired at two time points in the late evening (i.e., at 22:00h and 23:00h) to derive measures of the mean evening sAA levels. The use of late evening sAA levels yields the opportunities to study sAA at its peak values of the diurnal secretion pattern (Nater and Rohleder, 2009). This decreases the effects of measurement error and the episodic nature of sAA release, and reduces the potential influence of confounding by carbohydrate consumption and physical activity (Rohleder and Nater, 2009). In this article, we report on the associations between use of antidepressants, major depressive disorder and evening sAA.
major depressive disorder (MDD), and persons with current MDD. Control participants were defined as having no lifetime history of depressive disorder (MDD or dysthymia) or anxiety disorder (panic disorder, generalized anxiety disorder, agoraphobia, or social phobia) as assessed by the DSM-IV Composite International Diagnostic Interview (CIDI) version 2.1, no family history of depression, and a score below 14 on the 30-item Inventory of Depressive Symptomatology, Self-Report (IDS-SR) (Rush et al., 1996). These criteria were fulfilled by 413 respondents from the Netherlands Study of Depression and Anxiety respondents. Persons with remitted MDD (n ¼810) had a history of MDD but no diagnosis of MDD in the past 6 months as diagnosed by the CIDI. The third MDD group consisted of 1115 participants with a current diagnosis of MDD, i.e., in the past 6 months, as assessed by the CIDI (of whom 802 [71.9%] had been diagnosed with MDD in the past month). Of these participants, 1692 (72.4%) (329 control participants, 611 participants with remitted MDD, and 752 participants with current MDD) returned saliva samples (see below). Respondents on saliva collection did not differ from non-respondents in gender, but were older (age, 44.1 vs 38.2 years, respectively; Po 0.001), had a higher level of education (12.4 vs 11.6 years of education, respectively; Po 0.001) and less likely to be currently depressed (44.2% vs 57.4%, respectively; Po 0.001). Prior to participation, written informed consent was obtained from all participants. The study was approved by the ethics committee of the Leiden University Medical Center.
2.2. Salivary alpha-amylase At the baseline interview participants were instructed to collect saliva samples at home on a regular (preferably working) day shortly after the interview. The median time between the interview and saliva sampling was 9 days (25th–75th percentile, 4–22 days). Instructions prohibited eating, smoking, drinking, or brushing teeth within 15 min. Saliva samples were obtained using Salivettes (Sarstedt AG and Co, Nümbrecht, Germany) at two time points. Two evening samples were collected at 22:00 h and 23:00 h. Samples were stored in refrigerators and returned by mail. After receipt, salivettes were centrifuged, aliquoted, and stored at −80 1C. For assaying, after overnight thawing of the saliva at 4 1C, samples were diluted 50-fold with a Hamilton Microlab 500 B/C dilutor in physiological saline solution (Versylenes Fresenius, Cat. Nr. B230551). Analyses were performed using a kinetic colorimetric assay for total amylase activity (Cat Nr. 03183742, Roche Diagnostics, Mannheim, Germany) on a routine clinical chemistry analyzer. The total amylase assay is standardized to the IFCC reference measurement procedure (Lorentz, 1998), guaranteeing worldwide comparability of the data. Amylase activities are measured and expressed in IU/L at 37 1C. Within-run imprecision for the control pool ranged from 0.7% to 2.7% for the combined predilution step and the analysis across the entire study period. Between-run analytical imprecision was lower than 5% throughout this study.
2.3. Covariates We previously described significant determinants of sAA evening levels, i.e. age and alcohol intake (Veen et al., 2012). These identified determinants were considered as covariates in the present study. Alcohol consumption was expressed as number of units of alcohol per day. In addition we added gender as covariate.
2.4. Antidepressant use Medication use at the baseline assessment was collected via subject self-report (7 19%) and medication containers ( 7 81%) brought to the interview. Using the Anatomical Therapeutic Chemica (ATC) classification (www.whocc.no/atcddd/), psychoactive medication was categorized into antidepressants (tricyclic antidepressants [N06AA], selective serotonin reuptake inhibitors [N06AB], and other antidepressants [N06AF, N06AG, N06AX]), and benzodiazepines (N03AE, N05BA, N05CD, N05CF).
2. Method 2.5. Statistical analysis 2.1. Participants Data are from the Netherlands Study of Depression and Anxiety, a large cohort study on the course of depressive and anxiety disorders. In total, 2981 respondents were recruited from the community, general practice care, and specialized mental health care. The study sample included persons with psychopathological findings as well as control participants without a psychiatric diagnosis. For objectives and methods of the Netherlands Study of Depression and Anxiety, see the article by Penninx et al. (2008). The research protocol was approved by the ethical committee of participating universities, and all of the respondents provided written informed consent. To investigate whether sAA levels were different in depressed versus nondepressed respondents, taking into account remission of symptoms, three comparison groups (MDD status) were formed: control participants, persons with remitted
The distribution of sAA values was strongly positively skewed and therefore log-transformed values were used for analyses. Back-transformed geometric mean values are presented in tables and figures. On average, participants who provided both samples (n¼ 1659) showed higher sAA levels at 22:00 h compared to 23:00 h (t(1658) ¼12.0, po 0.001, r¼ 0.78). Because of the high correlation, the mean value of sAA levels at 22:00 h and 23:00 h was used in further analyses, to reduce the variability. When only one sample was provided (19 participants provided only a 22.00 h sample and 14 participants only a 23.00 h sample), that value was used. Baseline characteristics of the three MDD groups were compared using χ2 tests for categorical variables and one-way ANOVA for continuous variables. Multivariable analyses of covariance (ANCOVA) were used to examine differences in sAA levels across groups. All of the analyses were conducted using SPSS version 17.0 statistical software (SPSS Inc, Chicago, Illinois).
G. Veen et al. / Psychiatry Research 208 (2013) 41–46
3. Results 3.1. Clinical characteristics Participants were on average 44.3712.3 years old (range 18–65), and 66.5% were women. Characteristics across groups are presented in Table 1. Participants with remitted (n¼611) or current MDD (n¼752) were more often female and younger than controls (n¼ 329). Of the participants with current MDD, 210 (28.1%) were severely depressed as defined by an IDS score of 39 or higher, corresponding to a score of 20 or higher on the 17 item Hamilton Depression Scale. Of the participants with current MDD, 439 used no antidepressant, 28 were tricyclic antidepressant (TCA) users, 212 were selective serotonin reuptake inhibitor (SSRI) users and 73 used another antidepressant (3 duloxetine, 51 venlafaxine, 17 mirtazapine, 1 trazodone, 1 tranylcypromine). Of the participants with remitted MDD, 474 used no antidepressant, 18 were TCA users, 95 were SSRI users and 24 used another antidepressant (20 venlafaxine, 4 mirtazapine). Only one patient was treated with a non-selective monoamino-oxydase inhibitor (MAOI; tranylcypromine), and therefore its effects on sAA could not be ascertained in our study.
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persisted (p ¼0.06) (Table 2 and Fig. 1). When repeating the analyses in non-users of antidepressants no significant differences were found (Table 2). When using a continuous measure of depression severity (IDS) no association with sAA was found (r ¼0.02, p¼ 0.36). 3.3. Antidepressant use and salivary alpha-amylase In comparison to participants without antidepressants (221 U/ l; 95% CI: 211–232), users of TCAs showed significantly higher sAA levels (348 U/l; 95% CI: 271–447, p o0.001). After adjustment for age, gender, and alcohol intake these differences remained significant (p ¼0.003), even after additional adjustment for group (controls, remitted MDD, current MDD) (p ¼0.01) (Table 3 and Fig. 1). Users of SSRIs and other ADs did not show changes in sAA levels compared to participants without antidepressants (respectively, p ¼0.12 and p ¼0.27). When comparing the 74 users of serotonin-norepinephrine reuptake inhibitors (SNRIs: subgroup of the other AD users) to the 1242 participants not using antidepressants in a sensitivity analysis, the sAA levels were not statistically significantly higher in the SNRI group in the adjusted model 2 (mean 263; 95% CI: 216–312 vs mean 224 U/L; 95% CI: 213–235: p¼ 0.12).
3.2. MDD status and salivary alpha-amylase In unadjusted analyses, the geometric mean sAA was slightly higher in current MDD (230 U/l; 95% confidence interval [CI]: 216– 245) compared to remitted MDD (221 U/l; 95% CI: 206–237) and controls (214 U/l; 95% CI: 195–235), but these differences in sAA levels did not reach significance. However, a trend toward significance (p ¼0.06) was found for sAA when comparing participants with current MDD with controls after adjustment for age, gender, and daily alcohol intake. After additional adjustment for antidepressant use, this trend that approached significance
4. Discussion The present study showed that users of TCAs had significantly higher sAA levels compared with participants not using antidepressants, which remained significant after adjustment for covariates, i.e., gender, age, alcohol intake and group (controls, remitted MDD, and current MDD). We are not aware of previous studies that investigated this association. Additionally, a trend toward significance was found with higher sAA levels in participants
Table 1 Characteristics according to diagnoses of depressive disorders in 1692 participants. Demographic variables
Controls
Remitted MDD
Current MDD
P
No. of participants Female (%) Age (years, mean, S.D.) Daily alcohol intake (in units) (mean, S.D.) Antidepressant use TCA (%) SSRI (%) Other (%) Benzodiazepine use (%)
329 59.3 47.6 (11.7) 1.1 (1.3)
611 70.4 44.9 (12.5) 1.0 (1.4)
752 60.5 42.2 (12.1) 0.9 (1.4)
0.003 o 0.001 0.08
– – – 2.4
2.9 16.2 4.3 12.4
3.7 28.2 10.5 23.0
0.02 o 0.001 o 0.001 o 0.001
Data are presented as means 7 standard deviation (S.D.) or percentage. P-values of one-way ANOVAs and Chi-Square tests are presented. Abbreviations: MDD¼ major depressive disorders; TCA ¼ tricyclic antidepressant; SSRI ¼ selective serotonin reuptake inhibitor.
Table 2 Salivary alpha-amylase levels in controls, remitted and current MDD patients.
All participants No. Unadjusted mean (U/ml) Adjusted mean (Model 1) (U/ml) Adjusted mean (Model 2) (U/ml) Only non-users of antidepressants No. Unadjusted mean (U/ml) Adjusted mean (Model 1) (U/ml)
Controls
Remitted MDD
Pa
Current MDD
Pa
329 214 (195–235) 209 (190–230) 208 (189–230)
611 221 (206–237) 220 (206–236) 220 (206–236)
0.59 0.38 0.36
752 230 (216–245) 233 (219–248) 234 (219–249)
0.20 0.06 0.06
329 214 (195–235) 209 (190–230)
474 234 (207–242) 224 (207–241)
0.46 0.24
439 223 (206–242) 228 (211–248)
0.50 0.13
Data are presented as back-transformed geometric estimated means with 95% confidence intervals within groups. Pa: simple contrast p-value of patient group vs controls are presented. Model 1: adjusted for age, gender, and alcohol intake. Model 2: Additionally adjusted for kind of antidepressants (no antidepressants, TCA, SSRI, and other antidepressants).
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(Tulen et al., 1996) with current depression compared to control participants, but only after adjustment for covariates, i.e., gender, age and alcohol intake. Our findings consistent with those from Licht et al. (2008), who found that TCA use was strongly associated with ANS measures, e. g. heart rate variability, heart rate, blood pressure, and pre-ejection period. Some supporting findings were also found in animal data. In four studies, effects of TCAs were studied on salivary glands and oral microbiota in rats. These studies showed that there was a stimulating effect on the biosynthesis and secretion of alphaamylase (Koller et al., 2000,2001;Yu, 1992;Yu et al., 1989). This is in line with our findings. In another study the effects of salivary components of the tricyclic antidepressant imipramine (among other antidepressants) were studied. In this study sub-acute and chronic treatments of rats with the tricyclic antidepressant imipramine did not affect total protein and amylase levels (Kopittke et al., 2005. We did not find significant associations with the use of SSRIs and other antidepressants. In addition, other studies have also shown that the sympathetic part of ANS activity is higher in subjects using TCA than non-users (Barton et al., 2007; Lederbogen et al., 2001; Licht et al., 2010; van Zyl et al., 2008; Veith et al., 1994). The putative enhancing effects of TCAs on
Evening Salivary Alpha-Amylase (U/L)
400 380 360 340 320 300
P=0.06
280 260
P=0.36
240 220 200
P=0.01 P=0.12
0
=9 7
TC
A
A o
Cu r
N
us n= e SS 46 R Iu s n= e O th 30 7 er AD us n e
n= D 1. 24 2
52
=7
D n= D 61 1 re nt M D D n
9
M ed
em itt R
C
32
on t ro n= ls
P=0.27
Fig. 1. Salivary alpha-amylase across groups and kind of antidepressants on a logarithmic scale. The size of each square is proportional to the number of participants. Vertical lines indicate standard errors. P-values of multivariable analyses of covariance (ANCOVA) are presented after adjustment for age, gender, alcohol intake, and antidepressants use or group. Abbreviations: MDD ¼major depressive disorder; AD¼ antidepressants; TCA ¼ tricyclic antidepressants; SSRI¼ selective serotonin reuptake inhibitor.
sympathetic activity may have confounded the reported relationships between ANS and depression and anxiety in previous studies (Carney et al., 2005; Martens et al., 2008), which found elevated levels of plasma and urinary catecholamines, higher heart rate, and lower heart rate variability. A possible explanation for the effect of TCAs on ANS is that TCAs mainly inhibit the reuptake of norepinephrine, which increases its levels not only in the brain but also in the systemic circulation. Through accentuated antagonism norepinephrine has a blocking effect on muscarinic receptors, decreasing parasympathetic activity. In addition, the intrinsic anticholinergic effects of TCAs may even further inhibit parasympathetic activity (Esler et al., 1985; Manabe et al., 1991). In conclusion, lowered parasympathetic activity and increased sympathetic activity might be an effect induced by TCAs. We did not find convincing associations between sAA and depression, but only an association of borderline significance between participants with current MDD and controls. And when comparing MDD nonusers of antidepressants and controls no significant differences in sAA levels were found. Previous studies have shown mixed results. In studies that assessed sympathetic activity in depression employing non-cardiac specific measures such as norepinephrine spillover and skin conductance levels have yielded variable results. Most of these studies reported moderate increases in sympathetic activity in depression (Esler et al., 1985; Koschke et al., 2009; Veith et al., 1994), although some studies did not reveal any relationship (Barton et al., 2007; Guinjoan et al., 1995). Several studies suggest that beta-adrenergic activity or number of beta-receptors is decreased in depression (Bruno et al., 1983; Girdler et al., 1993; Krittayaphong et al., 1996; Light et al., 2009). This means that an increased sympathetic drive may be hidden by a decrease of effectiveness of the target organs to respond to the adrenergic drive. Some limitations should be recognized. First, sAA was assessed only in the evening, and therefore no statements can be made about associations between morning sAA and depression. Second, this study precludes drawing conclusions about whether sAA alterations in depression are more pronounced in total output (for which evening samples might be good proxy) or in change in daily rhythm. Multiple days of sampling may be necessary to reliably measure biological markers of the stress system (Hellhammer et al., 2007), but this was not an option in our large-scale study. Third, saliva for the assessment of sAA was collected using salivettes and participants were instructed to chew on the cotton roll, while active chewing rather than a passive drool may alter sAA (DeCaro, 2008). However, stress-induced increase of sAA levels has been shown to be independent of salivary flow rate (Rohleder et al., 2006). Fourth, because these analyses were crosssectional, our results do not indicate any causal directions of the associations found. Therefore, the higher sAA levels might be a consequence of depression or the treatment of depression. Our findings with respect to antidepressants might point in this
Table 3 Salivary alpha-amylase levels in non-users of AD, and in TCA, SSRI, and other AD users.
No. of participants Unadjusted mean (U/l) Adjusted mean (U/l) (Model 1) Adjusted mean (U/l) (Model 2)
No AD
TCA
Pa
SSRI
Pb
Other AD
Pc
1242 221 (211–232) 222 (211–233) 224 (213–235)
40 348 (271–447) 326 (254–418) 319 (248–409)
o 0.001 0.003 0.01
307 210 (191–231) 210 (190–231) 205 (185–226)
0.36 0.30 0.12
97 258 (217–307) 255 (215–302) 248 (208–295)
0.09 0.13 0.27
Data are presented as back-transformed geometric estimated means with 95% confidence intervals within groups. Pa: simple contrast p-value of TCA users vs non antidepressant users are presented. Pb: simple contrast p-value of SSRI users vs non antidepressant users are presented. Pc: simple contrast p-value of other AD users vs non antidepressant users are presented. Model 1: adjusted for age, gender, and alcohol intake. Model 2: additionally adjusted for group (controls, remitted MDD, current MDD). Abbreviations: MDD¼ major depressive disorders; TCA ¼ tricyclic antidepressant; SSRI ¼ selective serotonin reuptake inhibitor; AD ¼ antidepressant.
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direction. Fifth, there is still some controversy whether sAA is a valid and reliable measure of sympathetic activity of the ANS. Although there is growing evidence (Nater et al., 2005; Rohleder et al., 2006; Thoma et al., 2012), some issues remain unanswered, such as the relationship between salivary flow rate and parasympathetic activation, and other methodological aspects (see comments by Bosch et al. (2011)). Sixth, we did not have information on how long non-treated MDD patients did not take antidepressants and whether the two groups of patients (remitted vs current MD) differed in this variable. Lastly, although we assessed that depressed participants in our study had generally higher levels of stress and mood symptoms – as illustrated by higher IDS scores – compared with control participants, day-to-day variation that we could not further address could play an additional role in ANS function. Despite these limitations, our study had many strong aspects, including the large sample size with clearly distinct depression groups, the use of a recently introduced new measurement of ANS function, namely sAA, and the adjustment of various covariates. We conclude that higher sAA levels in depressed patients, indicative of an increased sympathetic activity, may be induced by TCAs. In addition, prospective studies are needed because of the high between-person variability. By using prospective study designs, within-patient comparisons over time of sAA levels would substantially increase the statistical power.
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