Salivary, total plasma and plasma free cortisol in panic disorder

J Neural Transm (2000) 107: 831–837

Salivary, total plasma and plasma free cortisol in panic disorder D. Wedekind, B. Bandelow, A. Broocks, G. Hajak, and E. Rüther Department of Psychiatry, University of Göttingen, Federal Republic of Germany Received July 28, 1999; accepted October 26, 1999

Summary. Background. Research on basal HPA axis activity in patients with panic disorder showed inconsistent results. Methods. Basal total plasma, plasma free and salivary cortisol levels were compared in patients with panic disorder (n 5 47) and in healthy individuals (n 5 23). Correlations between these fractions were calculated. Results. All three basal cortisol fractions were significantly elevated in patients compared to controls. There were significant correlations between all three cortisol fractions. Conclusions. Nonsignificant differences between cortisol levels of patients and healthy controls in previous studies may have been due to inclusion of less severely ill patients or to small sample sizes (96 words). Keywords: Panic disorder, cortisol, saliva, plasma, HPA axis. Introduction

Anxiety disorders have been associated with numerous claims of brain dysfunction, but the neurobiology behind the development of acute anxiety or panic attacks remains largely unclear. Of the many reported findings, several lines of evidence suggest that the hypothalamic-pituitary-adrenal (HPA) axis is disturbed in patients with panic disorder (PD). Measurements of total plasma cortisol, as well as assessment of free cortisol in plasma, saliva and urine have been done on patient populations of different sample size, different severity of illness and at different times and periods of the day. However, up to now the data have been inconclusive. Baseline total plasma levels of cortisol in PD patients were reported to be elevated in three studies and to be normal in eight studies (Table 1). Mostly the studies that failed to detect significant differences used rather small sample sizes. One study (Hollander et al., 1989) found elevated baseline cortisol levels only in a subgroup of patients who developed panic attacks after lactate infusion relatively late. In another investigation, differences between patients and controls were only found during the night and in patients who entered the study through clinical referral channels and not in those

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recruited by advertisements (Abelson and Curtis, 1996b). The authors regard this subgroup difference as a measure of illness severity, although objective criteria were not met. Studies investigating chemically provoced panic attacks showed conflicting results, too. While most studies regarding cortisol excretion during lactate induced (Carr et al., 1986; Den Boer et al., 1989; Gorman et al., 1989; Levin et al., 1987; Ströhle et al., 1998; Targum, 1992) and carbondioxide induced (Gorman et al., 1988; Woods et al., 1988; Gorman et al., 1989) panic attacks failed to discover changed cortisol levels, studies regarding yohimbine – e.g. (Charney et al., 1987a), M-chlorophenylpiperazine – (Charney et al., 1987b; Klein et al., 1991) or cholecystokinine-terapeptide(CCK-4) (Shlik, 1997; Depot, 1999) induced panic attacks revealed significant rises in cortisol excretion in patients with panic disorder. During spontaneous panic attacks, a subtle but significant increase in salivary cortisol levels was found (Bandelow et al., 1999). More than 90% of the cortisol is bound to corticoid-binding globulin (CBG), the remainder circulates freely (Kirschbaum and Hellhammer, 1989). Although only the free fraction is biologically active (Robbins and Rall, 1957), in the above cited investigations, only the total (CBG-bound and free) cortisol fraction was measured. Accordingly, other authors investigated the urinary secretion of cortisol which is a good measure of circulating free cortisol (Table 1). Urinary free cortisol (UFC) in PD patients was found to be normal in one study with a small sample size and elevated during the night in two studies. In the study by (Lopez et al., 1990), UFC was only elevated in patients with complicated PD (those with agoraphobia and secondary depression). In another study, UFC was lower in PD patients than in depressed patients and equal to schizophrenic and post-traumatic stress disorder patients, but a comparison with normal subjects was not made (Yehuda et al., 1993). Salivary cortisol concentration has been shown to reflect the biologically active plasma unbound cortisol level (Riad-Fahmy et al., 1982). Excellent correlations between salivary and free plasma cortisol have been reported in previous studies. Unless free cortisol is measured, the assessment of salivary cortisol was supposed to be the “better measure of adrenal cortical function than plasma cortisol” (Vining et al., 1983). In contrast to venepuncture, stressinduced influences can be neglected. Moreover, the rather complex separation procedure of free from CBG-bound cortisol can be substituted by saliva sampling. In a study on daytime salivary cortisol levels, only the more severely ill patients [as defined by a score of $22 on the Panic and Agoraphobia Scale; (Bandelow, 1995)] had significantly higher levels than the controls. In the present paper, total, free plasma and salivary basal cortisol secretion of PD patients in a comparatively large and more severely ill population were compared to healthy controls in order to further elucidate possible deviations of HPA axis function in PD. Methods The study design was approved by the institutional ethics committee. Subjects were consecutively admitted patients (n 5 47) of an outpatient anxiety disorders unit with

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Table 1. Studies on basal plasma and urinary free cortisol levels Author Plasma cortisol Nesse et al. (1984) Roy-Byme et al. (1986) Goldstein et al. (1987) Hollander et al. (1989) Abelson and Curtis (1996a) Liebowitz et al. (1985) Villacres et al. (1987) Cameron et al. (1987) Stein and Uhde (1988) Woods et al. (1988) Gurguis et al. (1991) Brambilla et al. (1992) Brambilla et al. (1995) Urinary free cortisol Bandelow et al. (1997) Bandelow et al. (subm) Lopez et al. (1990) Uhde et al. (1988) Salivary cortisol Bandelow et al. (subm)

n Patients

n Controls

Basal cortisol levels

14 8 24 103 20 20 10 8 10 14 32 17 12

6 30 61 32 12 17 10 4 10 8 12 14 12

≠ ≠ ≠ 5/≠a 5/≠b 5 5 5 5 5 5 5 5

16 23 66 12

13 23 62 12

≠ ≠ 5/≠c 5

23

23

5/≠d

elevated only in “late panickers”, b elevated only during the night in clinical referral patients, c elevated only in patients with agoraphobia and secondary depression, d elevated only in patients with P&A score $22 ≠ basal cortisol levels significantly elevated; 5 no significant differences a

panic disorder with or without agoraphobia according to DSM-IV (APA, 1994) and ICD-10 (WHO, 1991). Thirty-six patients (76.6%) had panic disorder with agoraphobia. Subjects were eligible when they had a severity score of at least 18 on the Panic and Agoraphobia Scale (clinician-rated version) (Bandelow, 1995). Exclusion criteria were severe medical illness, organic brain damage, history of psychiatric diseases, past or current major depression, unless it post-dated panic disorder, alcohol or drug abuse and pregnancy. Patients were free of psychotropic medication for at least one week before sampling. None of the patients showed symptoms of medication withdrawal. Twenty-three healthy volunteers were matched so that sex distribution (female patients: 66.0%, female controls: 69.6%; χ2-Test, p . 0.05) and mean age (patients 32.1; SD 8.1; controls: 34.3; SD 8.1; t-test; p . 0.05) did not differ significantly between patients and controls. Control subjects were screened for the absence of psychiatric disorders with the SCID [Structured Clinical Interview for DSM-III-R, (Wittchen et al., 1990)]. In order to control for diurnal cortisol variations, blood and saliva samples were all performed between 10:00 and 12:00 a.m. in patients and controls. Saliva was obtained with the “Salivette” sampling device (Sarstedt Inc., Rommelsdorf, Germany) which consists of a cotton swab on which the subject gently chews for 2 minutes. Thereafter, the swab is transferred into the inner plastic tube of the device. This tube has a little hole, allowing the saliva to run down into the outer tube during centrifugation. To stimulate saliva secretion, the cotton swabs contain citric acid. Patients were instructed to record whether they had drunk coffee or alcohol, had smoked, or had bleeding gums in the last hour

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before taking the saliva samples. As this could lead to changes in cortisol levels, such subjects were not included in the study.

Cortisol assay Blood samples were centrifuged after clotting and the supernatant was stored at 220°C. For measurement of total plasma cortisol levels 50 µl of plasma were extracted in 450 µl of ethanol. The organic phase was evaporated to dryness and redissolved in 1 ml of RIA buffer. 100 µl aliquots were used for the SPA-RIA. Salvia samples were stored at 220°C immediately after collection and thawed before analysis. After centrifugation 150 µl of saliva were used for duplicate analysis by radioimmunoassay (RIA). 150 µl of saliva were extracted into 1,350 µl of ethanol. The organic phase was evaporated to dryness and finally redissolved in 300 µl RIA buffer. Aliquots of 100 µl were used for duplicate RIA measurements using the scintillation proximity assay (SPA system Amersham-Buchler, Braunschweig, Germany) and tritium labelled cortisol (Biogenesis, Poole, UK). Recovery values average 70–80%. The sensitivity of the assay was 130.01 nmol/L. The interassay variance was less than 10%. For the separation of plasma free cortisol, thawed plasma samples were ultrafiltrated using the centrifree micropartition system (Amicon, Beverly, MA, USA). 50 µl of the filtrate were extracted into 450 µl of ethanol. The organic phase was evaporated to dryness, redissolved in 1 ml RIA buffer and assayed by the SPA-RIA. Basal cortisol levels and age differences were compared with the t-test for independent samples. A χ2-test was done for differences in sex distribution. Spearman rank correlations were calculated between total, free plasma and salivary basal cortisol. Tests were done using the Statistical Analysis system (SAS 6.12; SAS Institute, Heidelberg, Germany).

Results

The basal cortisol values are shown in Table 2. CBG-bound, free and salivary cortisol values were significantly elevated in patients compared to controls. All fractions correlated significantly with each other (Table 3). The highest correlations were found between plasma free and salivary cortisol. The severity of illness in our patients was higher than in a normative sample of 452 unselected panic patients (Bandelow, 1999) (Mann-Whitney-U-Test, U 5 3,006.5; p , 0.01). Discussion

In this study basal cortisol levels as assessed by salivary, plasma free and total levels were elevated significantly in PD patients. Though the majority of Table 2. Basal cortisol values in plasma (nmol/l); t-test for independent samples

Plasma total cortisol Plasma free cortisol Salivary cortisol

Patients (n 5 47)

Controls (n 5 23)

mean (SD)

mean (D)

515.7 (322.6) 11.24 (6.75) 10.82 (8.27)

364.9 (164.5) 7.22 (3.46) 6.80 (3.45)

t

p

2.59 3.29 2.81

,0.05 ,0.01 ,0.01


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Table 3. Spearman rank correlations between different cortisol fractions Fraction Patients (n 5 47) Plasma total cortisol Plasma free cortisol Controls (n 5 23) Plasma total cortisol Plasma free cortisol All subjects (n 5 70) Plasma total cortisol Plasma free cortisol a

Plasma free cortisol

Salivary cortisol

0.40b

0.45b 0.88c

0.62b

0.51a 0.86c

0.47c

0.49c 0.89c

p , 0.05, b p , 0.01, c p , 0.0001

previous studies measuring the basal levels of total plasma cortisol (CBGbound plus free) failed to show significant differences between patients and controls, our findings together with some other investigations showing elevated cortisol levels suggest that patients with a lower degree of severity were included in the studies not showing a difference or the sample sizes were too small to detect these differences statistically. In our study, only patients with a score of 18 or greater on the Panic and Agoraphobia Scale were included, and a relatively large sample size was chosen. The study indicates that HPA axis activity is increased in patients suffering from moderate or severe panic disorder under baseline conditions compared to healthy controls. Significant correlations were found between total plasma, plasma free and salivary cortisol for both PD patients and healthy controls. The advantages of the non-invasive measurement of salivary cortisol warrant the use of this method in future measurements of cortisol in psychiatric patients. References Abelson JL, Curtis GC (1996a) Hypothalamic-pituitary-adrenal axis activity in panic disorder – 24-hour secretion of corticotropin and cortisol. Arch Gen Psychiatry 53: 323–331 Abelson JL, Curtis GC (1996b) Hypothalamic-pituitary-adrenal axis activity in panic disorder: prediction of long-term outcome by pretreatment cortisol levels. Am J Psychiatry 153(1): 69–73 APA (1994) Diagnostic and statistical manual of mental disorders, 4th edn. American Psychiatric Press, Washington DC Bandelow B (1995) Assessing the efficacy of treatments for panic disorder and agoraphobia. II. The Panic and Agoraphobia Scale. Int Clin Psychopharmacol 10(2): 73–81 Bandelow B (1999) Panic and Agoraphobia Scale (PAS). Hogrefe, Göttingen Bern Toronto Seattle Bandelow B, Sengos G, Wedekind D, Huether G, Broocks A, Hajak G, Rüther E (1997) Urinary cortisol, testosterone, melatonin, and catecholamines in panic disorder. Pharmacopsychiatry 30: 1–5 Bandelow B, Wedekind D, Pauls J, Broocks A, Hajak G, Rüther E (2000) Salivary cortisol in panic attacks. Am J Psychiatry (in press)

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