CRH and GHRH test results to the outcome of depression – Preliminary results suggest the GHRH test may predict relapse after discharge

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JOURNAL OF PSYCHIATRIC RESEARCH

Journal of Psychiatric Research 42 (2008) 356–364

www.elsevier.com/locate/jpsychires

Relationships of DEX/CRH and GHRH test results to the outcome of depression – Preliminary results suggest the GHRH test may predict relapse after discharge Toshimi Owashi

a,b,*

, Tempei Otsubo a, Akihiko Oshima c, Kazuyuki Nakagome d, Teruhiko Higuchi e, Kunitoshi Kamijima a,f

a Department of Psychiatry, Showa University School of Medicine, Tokyo, Japan Section of Molecular Psychology, Max-Planck-Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany c Department of Psychiatry and Human Behavior, Gunma University Graduate School of Medicine, Gunma, Japan Division of Neuropsychiatry, Department of Multidisciplinary Internal Medicine, Tottori University Faculty of Medicine, Tottori, Japan e National Center of Neurology and Psychiatry, Musashi Hospital, Tokyo, Japan f International University of Health and Welfare, Tochigi, Japan b

d

Received 16 November 2006; received in revised form 17 January 2007; accepted 22 January 2007

Abstract To explore and compare hypothalamic–pituitary–somatotropic (HPS) axis function and hypothalamic–pituitary–adrenocortical (HPA) axis function in depression, the dexamethasone (DEX)/CRH test and growth hormone releasing hormone (GHRH) test were prospectively performed on patients with depression at the time of admission and discharge. The patients who relapsed within six months after discharge exhibited significantly lower growth hormone (GH) responses to GHRH at the time of discharge than those who did not relapse. There were no significant correlations between GH response to GHRH and the results of DEX/CRH tests after controlling for age, sex, and body mass index. The findings of this study suggest that results of the GHRH test may be a predictor of future relapse in patients with depression. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Hypothalamic–pituitary–adrenocortical (HPA) system; Hypothalamic–pituitary–somatotropic (HPS) system; Dexamethasone/corticotropine releasing hormone (DEX/CRH) test; Growth hormone releasing hormone test; Biomarker

1. Introduction Numerous studies have demonstrated abnormalities of the hypothalamic–pituitary–adrenocortical (HPA) system in major depression (Plotsky et al., 1998; Holsboer, 2000). The combined dexamethasone (DEX)/corticotropin releasing hormone (CRH) test is known to be one of the most sensitive means of assessing the HPA system (Heuser et al., 1994a,b; Oshima et al., 2000), and the results of the DEX/CRH test appear to be a state-dependent marker of * Corresponding author. Address: Section of Molecular Psychology, Max-Planck-Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany. Tel.: +49 89 30622 506; fax: +49 89 30622 605. E-mail address: [email protected] (T. Owashi).

0022-3956/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2007.01.006

depression (Kunugi et al., 2006) and to predict the individual likelihood of response to current antidepressant treatment (Ising et al., 2005, in press). There have also been studies of the hypothalamic–pituitary–somatotropic (HPS) axis in depression. Abnormal responses of growth hormone (GH) to growth hormone releasing hormone (GHRH) have also been reported in depression, though findings concerning this are not conclusive. Some studies have reported blunting of GH responses to GHRH in depression compared with controls (Eriksson et al., 1988; Lesch et al., 1989; Peabody et al., 1990; Dahl et al., 2000). Adolescents at high risk for depression as determined by family history also secreted significantly less GH than low-risk healthy controls on the

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GHRH test (Birmaher et al., 2000), similar to the case of healthy probands with a family history of mood disorder, who exhibited abnormalities on the DEX/CRH test (Holsboer et al., 1995). On the other hand, some other studies have reported that GH response to GHRH was significantly higher in patients with depression than in control subjects (Krishnan et al., 1988), or that no significant difference existed between patients with depression and control subjects in this response (Thomas et al., 1989; Brambilla et al., 1994). For instance, Gann et al. reported that responses to GHRH were not significantly lower in depressed patients than in controls (Gann et al., 1995). Although Dahl et al. reported that low GH response on the GHRH test in depression is maintained after reaching clinical remission (Dahl et al., 2000), it is still unclear what the results of GHRH test reflect. The differences in GH responses to GHRH among studies may be due to small sample sizes and differences in clinical characteristics of depression. Regarding sample size, most previous studies using GHRH test included fewer than 20 subjects with depression. Previous studies on the relationship between the HPS and HPA axes in depression have produced inconsistent results. Mokrani et al. reported that GH response to clonidine was lower in dexamethasone suppression test (DST) nonsuppressors than in suppressors (Mokrani et al., 1997). However, Lesch et al. found no relationship between GH responses to GHRH and ACTH and cortisol secretion following CRH administration without dexamethasone pretreatment (Lesch et al., 1989). Approximately 50% of patients with major depressive disorder (MDD) experience recurrence, and relapse or recurrence rates are as high as 20–37% during the continuation or maintenance phase of pharmacotherapy (Crown et al., 2002). Prevention of relapse is therefore one of the most important and challenging goals in the management of major depression. The findings noted above suggest that both HPA axis (Zobel et al., 2001) and HPS axis (Coplan et al., 2000) parameters may have the potential to predict future episodes of depression. To our knowledge, no longitudinal studies have compared HPA function measured by the DEX/CRH test and HPS function measured by the GHRH test in depression. To determine whether hormonal test results predict the prognosis of depression under the hypothesis that HPA dysfunction precedes poor outcome and that some relationship may exist between the HPS and HPA axes in depression, we prospectively performed two neuroendocrine challenge tests, the DEX/CRH test and GHRH test, in individuals with depression, and compared the results between those with and those without relapse within six months after discharge. 2. Methods 2.1. Subjects This was a prospective study performed in a natural clinical setting without performance of sex- or age-match-

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ing in advance. The participants were 33 inpatients (25 women and 8 men), mean age at admission: 57.4 years (standard deviation (SD) = 14.8) of the Department of Psychiatry, Showa University School of Medicine, who were diagnosed with MDD or dysthymic disorder with previous major depressive episode(s) according to the criteria of the Diagnostic and Statistical Manual for Mental Disorders 4th Edition (DSM-IV)(APA, 2000). Exclusion criteria were somatic disorders such as inflammation, endocrine disorders, or neoplasm, treatment with liverenzyme-inducing drugs, such as phenytoin, rifampicin, or carbamazepine, history of dependence or abuse of illicit drugs or other substances such as benzodiazepines and alcohol, shift workers, and pregnant women. All patients were biologically unrelated Japanese individuals. One male subject had dysthymic disorder with a previous major depressive episode, two women were diagnosed as having MDD on admission but their diagnoses were changed to bipolar II disorder after detailed information on the course of illness was obtained from their families, and 30 were diagnosed with MDD. The two patients whose diagnoses were changed to bipolar II before discharge were excluded from analysis in this study. Twelve participants (38.7%) had a family history of mood disorder in first- or second-degree relatives. Family history was confirmed using the Family History-Research Diagnostic Criteria. Mean number of previous episodes of major depression was 2.1 (SD = 1.1). The mean duration of hospitalization of the subjects was 75.3 (SD = 44.9) days. Mean socioeconomic status (Hollingshead, 1975) was 2.9 (SD = 0.72). All of the subjects but those with bipolar II disorder underwent pharmacological therapy; 16 with selective serotonin reuptake inhibitors, 8 with serotonin-noradrenaline reuptake inhibitors, 5 with tricyclic antidepressants, 1 with lithium carbonate and 1 with valproate. One patient treated with lithium carbonate and one with valproate did not use antidepressants because of previous side effects or lack of efficacy. Type of medication affected neither the results of hormonal tests nor relapse within six months after discharge on analyses with age, sex, and body mass index (BMI) as covariates. At discharge, two of the subjects with MDD could not be examined because of stroke or other physiological problems. Timing of discharge was decided mainly by the attending psychiatrists after consultations with the patient and/or the patient’s family. The severity of psychiatric disease was assessed with the 21-item version of the Hamilton Depression Scale (HAM-D) (Hamilton, 1967) and Global Assessment of Functioning (GAF) (APA, 2000) at the time of admission as well as at discharge. Relapse was considered to have occurred when the subjects met the criteria for MDD again after discharge, and/or required hospitalization for depression. This study was approved by the Institutional Review Board of Showa University, and all the participants submitted written informed consent.

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2.2. Hormonal tests The DEX/CRH test and GHRH test were performed twice on each patient, at the time of admission and at discharge. Either of the hormonal tests was performed at first, in random fashion, and the other hormonal test was performed within three to seven days. The first of the hormonal tests was performed within two to seven days after admission. The DEX/CRH test was performed as described in detail elsewhere (Heuser et al., 1994a; Holsboer et al., 1987; Zobel et al., 2001): 1.5 mg DEX was administered orally at 23:00, and an intravenous catheter with heparinized physiological saline was inserted into a forearm vein at 14:30 and 100 lg human CRH (hCRH ‘Mitsubishi’, Mitsubishi Pharma Corporation, Tokyo, Japan) was then injected intravenously at 15:00 immediately after the first (= 0 min) blood collection on the following day. Plasma ACTH and cortisol concentrations were measured at 30, 45, 60, and 75 min post-injection. In the GHRH test, the patients fasted beginning the previous night from 21:30 except for water and drugs. On the day of examination, an intravenous catheter with heparinized physiological saline was inserted into a forearm vein at 8:30, and 100 lg human GHRH (‘‘GRF Sumitomo 100’’; growth hormone-releasing factor (human)-(1-44)-peptide amide hexaacetate, Sumitomo Pharma Corporation, Tokyo, Japan) was injected intravenously at 9:00 immediately after the first (= 0 min) blood collection. Serum GH concentrations were measured at 15, 30, 60, and 90 min post-injection of GHRH. In both hormonal tests, subjects rested supine throughout the test in their hospital beds. Blood samples were cooled in ice water and immediately centrifuged (10 min at 3000 rpm at 4 °C) and stored at 20 °C. Plasma concentrations of ACTH and cortisol and serum concentration of GH were measured by radioimmunoassay at SRL Corporation (Tokyo, Japan). The limits of detection for ACTH, cortisol, and GH were 5.0 pg/ml, 1.0 lg/dl, and 0.05 ng/ ml, respectively. 2.3. Data presentation Areas under the concentration–time curve (AUC, arbitrary units) were calculated according to the trapezoidal rule (CortAUC for cortisol, ACTHAUC for ACTH, and GHAUC for GH). CortAUC was calculated from the Cort0, Cort30, Cort45, Cort60, and Cort75 values, ACTHAUC from the ACTH0, ACTH30, ACTH45, ACTH60, and ACTH75 values, and GHAUC from the GH0, GH15, GH30, GH60, and GH90 values. CortMAX indicates the maximum cortisol response to CRH. 2.4. Statistical analysis All statistical analyses were performed with SPSS for Windows (version 12.0.1, SPSS Inc., Chicago, USA). Pear-

son correlation analysis was performed to assess the relationships among age, BMI, number of previous episodes of depression, and HAMD and GAF at the time of admission and discharge. Partial correlation analysis was performed to assess the relationships between the results of the DEX/CRH test and GHRH test at the time of admission or discharge after controlling for age, sex, and BMI. Patients who relapsed within six months after discharge were classified as exhibiting relapse, while all other patients were classified as not exhibiting relapse. Demographic data, diagnoses, clinical features, and medications were compared between these groups with Pearson v2 or Fischer’s exact tests in case of qualitative data and with t-tests for independent samples in case of quantitative data. The Mann–Whitney U test was used for comparison of results of hormonal tests for assessment of differences by sex. The Wilcoxon signed-rank test was used for comparisons of the results of hormonal tests between the time of admission and discharge. Differences in AUCs of the hormonal tests between the relapse and non-relapse groups were evaluated by univariate analysis of covariance (ANCOVA) using age, sex, and BMI as covariates. Findings of p < 0.05 were considered significant. 3. Results 3.1. Demographic and clinical characteristics In 29 patients (22 women and 7 men, 57.4 (SD = 14.9, range 30–80) years) who could be evaluated during hospitalization, the scores of HAMD and GAF were significantly improved (paired t-test; p < 0.001): HAMD: from 26.6 (SD = 7.7) at the time of admission to 9.3 (SD = 4.8) at the time of discharge, and GAF from 38.6 (SD = 7.9) to 72.9 (SD = 9.9). There was a significant positive correlation between HAMD at the time of admission and age (Pearson correlation 0.401, p = 0.031) as well as a significant negative correlation between HAMD at the time of admission and BMI (Pearson correlation 0.379, p = 0.043). Women had significantly higher age, age at onset, HAMD score at the time of admission, and lower BMI than men (p < 0.05, see Table 1a). Within six months after discharge of the 29 patients, one patient with dysthymic disorder stopped visiting the hospital, one was hospitalized for physical problems, and one had been placed in a distant nursing home. There were no significant differences in demographic or clinical factors or results of hormonal test between the group that could be followed and the group that could not. Among the 26 subjects (20 women and 6 men, mean age: 55.9 (SD = 13.5) years) regularly followed for more than six months, six patients (4 women and 2 men) relapsed. Four of them relapsed within one month, one within two months, and the other patient within six months after discharge. Among the patients who relapsed, two of the four who relapsed within one month after discharge had obvious stressors (one had stress due to returning to work, one suffered the death of his

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father). They were also treated as patients who suffered relapse. Although no obvious stressors were detected in other patients, most patients with major depressive disorder are known to experience psychosocial burden. Four patients in the relapse group required hospitalization again for depression, while two patients had antidepressant doses increased. None of the subjects including those who relapsed after discharge, exhibited poor compliance/adherence to medication regimens according to the information obtained from the patients and their family and/or partners. 3.2. Changes in hormonal responses on DEX/CRH test and GHRH test On several occasions, the maximum values were obtained at the last point of measurement, suggesting that values might not have reached peak levels. At the time of admission, 4 ACTHMAX values were collected from ACTH75, 10 CortMAX values were from Cort75, and 6 GHMAX values were from GH90. At the time of discharge, 4 ACTHMAX values were collected from ACTH75, 5 CortMAX values were from Cort75, and 2 GHMAX values were from GH90. ACTHMAX on the DEX/CRH test was significantly lower at the time of discharge than at the time of admission (28.9 (SD = 35.5) at the time of admission to 18.2 (SD = 17.6) at the time of discharge, p = 0.042, Wilcoxon signed-rank test), but there were no significant differences in ACTHAUC or CortAUC on the DEX/CRH test or GHAUC on the GHRH test between the times of admission and discharge. 3.3. Relationships between results of hormonal tests and demographic or clinical characteristics Women had significantly higher levels than men on the DEX/CRH test at both the time of admission and that of

Table 1b Differences in results of hormonal tests by sex Women (N = 22)

Age (years) Age at onset (years) Family history Previous episodes Body mass index Duration of this hospitalization (days) HAMD-21 at admission HAMD-21 at discharge GAF at admission GAF at discharge Dose of antidepressants at discharge (equivalent to imipramine, mg/day)

p*

62.6 ± 12.3 55.0 ± 13.5 11 (50.0%) 2.1 ± 1.1 21.2 ± 3.8 81.1 ± 47.9

41.0 ± 10.0 36.1 ± 12.1 1 (14.3%) 2.3 ± 0.95 25.4 ± 4.8 57.0 ± 29.5

<0.001 0.003 0.187 0.680 0.022 0.223

28.3 ± 7.2 9.7 ± 5.0 37.6 ± 8.2 72.5 ± 9.4 142.6 ± 64.4

21.0 ± 6.8 7.9 ± 3.9 41.7 ± 6.8 74.3 ± 12.1 126.8 ± 55.2

0.025 0.378 0.239 0.678 0.585

Mean ± standard deviation. MDD, major depressive disorder; BP II, bipolar disorder type II; HAMD, Hamilton Depression Scale; GAF, Global Assessment of Functioning (DSM-IV). * p values (two-tailed) of exact Fisher tests(qualitative data) and t-test for independent samples (quantitative) are reported.

p*

29.1 ± 27.5 8.5 ± 7.8

8.3 ± 2.4 2.2 ± 0.51

0.009 0.020

15.7 ± 19.1

19.8 ± 17.5

0.389

At discharge DEX/CRH test ACTHAUC CortAUC GHRH test GHAUC

19.4 ± 14.5 7.3 ± 6.7

9.5 ± 3.1 1.9 ± 0.70

0.391 0.025

15.3 ± 14.4

19.6 ± 14.6

0.353

Mean ± standard deviation. * p values (two-tailed) of Mann–Whitney U test are reported.

discharge (p < 0.05, see Table 1b). Age exhibited significant positive correlations with ACTHAUC, ACTHMAX, CortAUC, and CortMAX at the time of admission and significant negative correlations with GHAUC at the time of admission and GHMAX at the times of admission and discharge (p < 0.05, see Table 2). There was a significant negative correlation between BMI and GHMAX at the time of admission (Spearman’ correlation 0.408, p = 0.035). After controlling for age, there was a stronger negative correlation between BMI and GHMAX at the time of admission (partial correlation coefficient 0.464, p = 0.017). After controlling for age, sex, and BMI, there were no significant relationships between the results of hormonal tests and severity of depression. However, HAMD at the time of admission exhibited a tendency toward positive correlations with CortAUC (partial correlation coefficient Table 2 Spearman’s correlations between results of hormonal tests and demographic and clinical characteristics Age

Men (N = 7)

Men (N = 7)

At admission DEX/CRH test ACTHAUC CortAUC GHRH test GHAUC

Table 1a Differences in demographic and clinical characteristics by sex Women (N = 22)

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At admission DEX/CRH test ACTHMAX ACTHAUC CortMAX CortAUC GHRH test GHMAX GHAUC At discharge DEX/CRH test ACTHMAX ACTHAUC CortMAX CortAUC GHRH test GHMAX GHAUC

BMI

Previous episodes

HAMD

GAF

0.370* 0.381* 0.396* 0.398*

0.042 0.021 0.119 0.152

0.019 0.018 0.001 0.044

0.465* 0.444* 0.480* 0.428*

0.363* 0.376* 0.423* 0.389*

0.319 0.410*

0.408* 0.310

0.250 0.261

0.159 0.306

0.295 0.360

0.136 0.059 0.236 0.345

0.147 0.101 0.202 0.158

0.171 0.219 0.106 0.051

0.058 0.019 0.072 0.042

0.083 0.163 0.227 0.216

0.461* 0.528*

0.388 0.377

0.222 0.243

0.274 0.311

0.380 0.399

BMI, body mass index; HAMD, Hamilton Depression Scale; GAF, Global Assessment of Functioning (DSM-IV). * p < 0.05 (two-tailed).

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Table 3 Demographic and clinical characteristics of patients with and without relapse within six months after discharge

Sex (women/men) Age (years) Age at onset (years) Family history Previous episodes Body mass index Duration of this hospitalization (days) HAMD-21 at admission HAMD-21 at discharge GAF at admission GAF at discharge Dose of antidepressants at discharge (equivalent to imipramine, mg/ day)

Relapse (N = 6)

Non-relapse (N = 20)

p*

4/2 56.0 ± 15.0 49.3 ± 14.7 3 (50.0%) 2.5 ± 1.4 25.5 ± 3.6 74.5 ± 75.0

16/4 55.9 ± 13.5 49.6 ± 14.4 7 (14.3%) 2.0 ± 1.0 20.8 ± 3.6 67.0 ± 24.3

0.596 0.988 0.969 0.644 0.342 0.010 0.692

24.3 ± 12.1 8.6 ± 3.4 36.7 ± 11.3 76.8 ± 12.3 115.8 ± 51.0

27.1 ± 7.6 8.7 ± 5.0 39.6 ± 7.7 74.5 ± 9.5 156.9 ± 58.2

0.512 0.967 0.468 0.618 0.136

Mean ± standard deviation. MDD, major depressive disorder; BP II, bipolar disorder type II; HAMD, Hamilton Depression Scale; GAF, Global Assessment of Functioning (DSM-IV). * p values (two-tailed) of exact Fisher tests(qualitative data) and t-test for independent samples (quantitative) are reported.

0.405, p = 0.055) and CortMAX (partial correlation coefficient 0.385, p = 0.070) at the time of admission. GAF at the time of admission also exhibited a tendency toward significant negative correlations with CortAUC (partial correlation coefficient 0.359, p = 0.093) and CortMAX (partial correlation coefficient 0.358. p = 0.094) at the time of admission. 3.4. Correlations between results of DEX/CRH test and GHRH test

Table 4 Results of DEX/CRH and GHRH tests in the relapse and non-relapse groups Non-relapse (N = 20)

F

p

At admission DEX/CRH test ACTHAUC 25.1 ± 22.8 CortAUC 10.1 ± 9.9 GHRH test GHAUC 7.7 ± 5.4

Relapse (N = 6)

25.1 ± 22.8 6.0 ± 5.2

0.388 0.915

0.540 0.350

19.5 ± 21.0

0.057

0.813

At discharge DEX/CRH test ACTHAUC 10.3 ± 8.0 CortAUC 5.7 ± 7.2 GHRH test GHAUC 3.8 ± 0.87

18.4 ± 11.6 6.0 ± 5.8

0.811 0.571

0.386 0.464

22.4 ± 13.2

4.999

0.044

Mean ± standard deviation, ANCOVA with covariates: age, sex, and body mass index.

age, age at onset, family history of mood disorder, number of previous episodes of depression, duration of hospitalization, or diagnosis before discharge between the relapse and non-relapse groups. Concerning severity of depression, there were no significant differences in HAMD or GAF at the time of admission or discharge between the two groups (Table 3). Fig. 1 illustrates the differences in serum GH response curves throughout the GHRH test at the time of admission and discharge between the relapse and non-relapse groups. The relapse group had significantly lower GHAUC at the time of discharge than the non-relapse group, after controlling for sex, age, and BMI (R2 = 0.365, F = 4.999, p = 0.044, see Table 4). 4. Discussion

In partial correlation analyses controlling for age, sex and BMI, no significant correlations were found between GH responses to GHRH and the results of DEX/CRH tests. 3.5. Comparison of findings for relapse and non-relapse groups

GH serum concentration (ng/ml)

The relapse group had higher BMI values than the nonrelapse group. There were no significant differences in sex,

We performed DEX/CRH and GHRH tests on inpatients with depression at the times of admission and discharge. Altogether, 23.1% of the patients that were followed relapsed within six months after discharge, and the relapse group exhibited significantly lower GH responses to GHRH at the time of discharge than the non-relapse group after controlling for age, sex, and BMI. These two groups did not differ

20 15 10

relapse: at admission relapse: at discharge GHRH

non-relapse: at admission 5 0

non-relapse: at discharge

0

15

30

60

90 (min)

Fig. 1. Mean serum concentration–time curve of GH response to GHRH test at admission (dotted line) and at discharge (solid line) for subjects with relapse (squares; N = 6) and non-relapse (circles; N = 20) within six months after discharge.

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in scores of HAMD or GAF at the time of admission or discharge, doses of antidepressants at the time of discharge, or demographic characteristics other than BMI. 4.1. Effects of age, sex, and BMI on the results of hormonal tests 4.1.1. Effects of age Effects of age and sex were noted in the DEX/CRH test and of age and BMI in the GHRH test in this study. Inconsistent findings have been obtained regarding the effects of age on the results of the DEX/CRH test. Kunzel et al. reported that there was no significant effect of age on hormonal responses to the DEX/CRH test in acutely depressed inpatients (Kunzel et al., 2003). In our subjects, however, there were significant positive correlations between AUC and peaks of both ACTH and cortisol at the time of admission, consistent with other previous reports that cortisol response to the DEX/CRH test increased with age in patients with depression (von Bardeleben and Holsboer, 1991; Heuser et al., 1994a). In our study, effects of age on the DEX/CRH test were observed at the time of admission, with no significant correlation noted between age and the results of the DEX/ CRH test at the time of discharge. The reason for this might have been that the magnitude of effects of age on the DEX/CRH test increases during major depressive episodes, although effects of age on hormonal responses have also been reported in healthy controls (Heuser et al., 1994a; Kudielka et al., 1999). GH responses to GHRH exhibited a significant negative correlation with age both at the time of admission and that of discharge in this study. This suggests that there was a consistent effect of age on results of the GHRH test in depressed subjects, as in healthy subjects, who exhibit an effect of aging on GH secretion (Arvat et al., 1997). In addition, the GHRH ‘‘GRF Sumitomo 100’’ has been found to yield lower GH responses in healthy subjects more than 30 years of age (Dainippon Sumitomo Pharma Co., Ltd.). Shibasaki et al. reported that somatotroph cells become less sensitive to GHRH with aging (Shibasaki et al., 1984). A recent animal study has also shown that the number of GHRH-ir neurons and the number and in size of GH-ir cells are significantly decreased in aged rats (Kuwahara et al., 2004). The results of DEX/CRH and GHRH tests in our study suggest the possibility that aging is associated with changes in HPA and HPS responses to hormonal tests in depressed patients. 4.1.2. Effects of sex Consistent with the many previous studies that have reported the finding of a sex difference in hormonal responses on the DEX/CRH test (Kunugi et al., 2006; Kunzel et al., 2003; Heuser et al., 1994a), in our sample hormonal secretion was higher in women than in men on

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the DEX/CRH test both at the time of admission and that of discharge. On the other hand, there are conflicting reports regarding the effects of sex on GH responses. Arvat et al., in a review, noted that GH-releasing effect is not dependent on sex, but does exhibit age-related variation (Arvat et al., 1997). The GHRH ‘‘GRF Sumitomo 100’’ used in this study has also been reported to exhibit no differences in results by sex among healthy subjects (Dainippon Sumitomo Pharma Co., Ltd.), and in the present study no difference was noted by sex in the results of GHRH tests in patients with depression. Our results thus suggest that HPS axis activity in depression might not be influenced by sex, though this is not the case for hormonal responses to the DEX/CRH test, representing the HPA axis. 4.1.3. Effects of BMI There was a negative correlation between BMI and GHMAX on the GHRH test at the time of admission. This finding is consistent with the above-mentioned report on the GHRH ‘‘GRF Sumitomo 100’’ that, among healthy individuals, those with a degree of obesity greater than +20% (equivalent to BMI > 24) exhibited low GH responses to GHRH (Dainippon Sumitomo Pharma Co., Ltd.). The results of the present study suggest that BMI affects the results of GHRH test in depressed patients as well as in healthy subjects. 4.2. Relationships between severity of depression and results of hormonal tests The findings of DEX/CRH test in the present study, that ACTHMAX at the time of discharge was significantly decreased compared with the time of admission and that some results of DEX/CRH test exhibited tendencies toward correlation with severity of depression and general function at the time of admission after controlling for age, sex and BMI, suggest the possibility that results of these tests can be considered a state marker, in accordance with previous studies (Rybakowski and Twardowska, 1999; Kunzel et al., 2003; Isogawa et al., 2005; Kunugi et al., 2006) that monitored HPA axis abnormalities in patients with depression during treatment. The lack of significant correlation between HAMD and the results of DEX/ CRH test at the time of discharge might have been due to the narrow between-subject variance in HAMD at the time of discharge. Some studies have reported that low GH secretion or GH responsiveness might be considered a trait marker in depression (Puig-Antich et al., 1984; Jarrett et al., 1990; Birmaher et al., 2000). Jarrett et al. reported that depressed patients exhibited significant reduction in GH secretion during sleep that persisted through treatment and recovery into the drug-free state of remission. In an animal study, Coleman et al. reported that behaviorally nonreactive monkeys had low GH responses to GHRH (Coleman et al., 2003). There appears to be inconsistency in findings

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regarding GH responsivity as a state marker for depression. While Coote et al. reported a change in GH response to clonidine pre- to post-treatment (Coote et al., 1998), Ryan et al. reported no association between severity of depression and GH secretion in adolescents with MDD (Ryan et al., 1988). The findings of our study suggest that it is unlikely that the GHRH test can be considered a state marker, since there were no significant correlations between GHAUC or GHMAX and HAMD or GAF after controlling for age, sex and BMI or after controlling for age and BMI, and no significant changes in GH responses to GHRH between the time of admission and discharge.

to treatment (Esel et al., 2004). However, it is difficult to conclude that the result of GHRH test is a persistent marker of depression, since in the present study there were no significant differences in GH responses to GHRH between the relapse and non-relapse groups at the time of admission. However, HPS abnormality represented by low GH response to GHRH might be related to liability to relapse and be a prognostic marker in patients with depression.

4.3. Relationship between outcome and results of hormonal tests

It has been suggested that corticosteroid–serotonin (5HT) interactions are central to the pathophysiology of depression. The 5-HT system and HPA axis appear to have complex inter-relationships, although no clear conclusions have been reached regarding the relationship between HPA axis function and 5-HT function in depression (Porter et al., 2004). GH response to clonidine and L-tryptophan (L-TRP) is reduced in depression, suggesting reduced a-2adrenergic and 5-HT1A receptor function (Watson et al., 2000). It is conceivable that 5-HT-mediated relationships exist between the HPA axis and HPS axis, since both appear to be related to the 5-HT system. Concerning the relationship between the HPA axis and HPS axis in depression, our findings support the report of Lesch et al., who found no correlation between them contrary to our hypothesis, although our methods differ from theirs in terms of both pretreatment with DEX and dose of GHRH (Lesch et al., 1989). Young et al. evaluated the correlation between clonidine-stimulated GH secretion and ACTH response to an emotional stressor in healthy controls and depressed patients, and found no significant correlation with either in the depressed patient group, although significant correlations were found in normal subjects (Young et al., 2005).

The results of previous studies regarding the possibility of using DEX/CRH test as a predictor of outcome of depression are confusing. Although some studies have reported that the responses of cortisol or ACTH to CRH in DEX/CRH tests were associated with intermediate-term relapse (Zobel et al., 2001; Hatzinger et al., 2002; Appelhof et al., 2006) and Ising et al. suggested that a second DEX/ CRH test is required for optimal prediction of lack of response to antidepressant treatment (Ising et al., 2005, in press), another study reported that cortisol response did not differ significantly between patients who did and those who did not obtain remission from bipolar disorder (Watson et al., 2004). In the present study, the results of DEX/ CRH test were not correlated with relapse within six months after discharge, suggesting that HPA axis parameters might not predict prognosis following hospital treatment of depression, though they might play roles as state-dependent markers. Although several studies have suggested the possibility that GH response is a risk factor for or trait marker of depression (Puig-Antich et al., 1984; Jarrett et al., 1990; Birmaher et al., 2000; Dahl et al., 2000), there is little clear evidence for GH response as a predictor of outcome of depression. There have been findings that decreased GH response to clonidine at baseline is correlated with treatment response (Coote et al., 1998; Correa et al., 2001), and Gann et al. observed significant correlations between GH responses to clonidine and GHRH in both patients with depression and healthy controls (Gann et al., 1995). In the present study, the results of GHRH test at the time of admission did not predict relapse after discharge. However, lower GH responses to GHRH test at the time of discharge predicted relapse within six months after discharge, while no other demographic and/or clinical indices but BMI differed significantly between the relapse and nonrelapse groups. The finding of lack of significant change in GH response to GHRH between the time of admission and that of discharge might reflect the trait-dependency of this test, as suggested by Esel et al., who reported that L-DOPA-induced GH responses did not change with treatment in depressed patients even if they exhibited a response

4.4. Relationship between the HPA axis and HPS axis in depression

4.5. Limitations Some limitations to this study must be mentioned. First, this study was limited in particular by its small sample, which might have given rise to type II errors. For example, although there was no significant difference in dose of antidepressants at the time of discharge between the relapse and non-relapse group, the possibility remains that insufficient doses of antidepressants led to relapse, since the sample size was too small to obtain significant differences between doses of antidepressants. Moreover, since we did not take into account multiplicity in statistical analyses, the present findings should be considered exploratory. In addition, plasma DEX concentrations prior to CRH injection in DEX/CRH test were not available, although subjects were confirmed to have taken DEX by the medical staff during hospitalization. There were also differences among patients in both the order and intervals between hormonal tests, since repeated per-

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formance of the two hormonal tests was required. However, since hospitalization itself can be a markedly stressful event for patients, we avoid performing hormonal tests on the first or second day after admission. To avoid effects of prior hormonal testing, we arranged a longer than three-day interval between the two tests. Since some maximum values in neuroendocrine tests were collected from the last point of measurement, it is unclear whether they represent true peak levels in neuroendocrine responses to hormone stimulation. Determination of compliance/ adherence to medication regimens was based on information supplied by the patients and their family and/or partners, though serum concentrations of antidepressants were not measured. Finally, medications received by subjects might have affected the results of hormonal tests, although we excluded subjects receiving carbamazepine, and no significant relationships were found between test results and types of antidepressants in this study, as in previous reports (Heuser et al., 1996; Correa et al., 2001; Kunzel et al., 2003; Kunugi et al., 2006). Moreover, Watson et al. (2004) reported that neither DEX levels nor delta cortisol differed between those taking and not taking CYP3A4-inhibiting drugs, lithium, or selective serotonin reuptake inhibitors. In conclusion, in the present study, DEX/CRH test is likely to be a state marker for depression, and the result of GHRH test at the time of discharge was found to predict relapse within six months after discharge for subjects with depression. Effects of age and BMI should be taken into account in assessing the results of GHRH tests and of age and sex in assessing those of DEX/CRH tests. Given the low R-squared value presumably due to the small sample size, further investigations are required to draw definitive conclusions regarding the relationship between HPS function and the outcome of depression. Role of funding source This study was supported in part by a grant from the Mitsubishi Pharma Corporation and by a grant from the Uehara Memorial Foundation. Both the Mitsubishi Pharma Corporation and the Uehara Memorial Foundation had no further role in study design; in the recruitment of the subjects; in the analysis and interpretation of data; in the writing of the paper; and in the decision to submit the paper for publication. Conflict of interest We declare that we have no conflicts of interest. Acknowledgements The authors wish to thank the medical staff of Showa University School of Medicine for assistance in conducting this study.

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