HUMAN PSYCHOPHARMACOLOGY
Hum. Psychopharmacol. Clin. Exp. 14, 447±451 (1999)
Serum Trace Elements in Animal Models and Human Depression. Part II. Copper MAèGORZATA SCHLEGEL-ZAWADZKA1*, ANDRZEJ ZIE˛BA2, DOMINIKA DUDEK2, MIROSèAW KROSÂNIAK1, MARIA SZYMACZEK2 and GABRIEL NOWAK3,4 1Department
of Food Chemistry and Nutrition, Collegium Medicum, Jagiellonian University, KrakoÂw, Poland Department of Psychiatry, Collegium Medicum, Jagiellonian University, KrakoÂw, Poland 3 Institute of Pharmacology, Polish Academy of Sciences, KrakoÂw, Poland 4 Laboratory of Radioligand Research, Collegium Medicum, Jagiellonian University, KrakoÂw, Poland 2
In the present study we report the results of investigations into the serum copper levels in a clinical study of 19 patients with unipolar depression; 16 normal controls and three animal models of depression: chronic severe stress (CSS), chronic mild stress (CMS) and olfactory bulbectomy (OB) in rats. Unipolar depressed patients exhibit signi®cantly higher serum copper levels than the appropriate controls (depression 1.15+0.17 mg/l; control 0.95+0.09 mg/l). There was no alteration in that value in rat models of depression. The data indicate that the increased serum copper level in the depressed patients might potentially be a marker of that illness. Moreover, animal models of human depression do not show changes in this marker. Copyright # 1999 John Wiley & Sons, Ltd. KEY WORDS
Ð serum; copper; depression; animal models; human
INTRODUCTION Interest in a copper role in depression is not widespread, although this element is an important component of enzymes that take part in the catecholaminergic pathways involved in the pathophysiology of depression ( for a review see Heninger et al., 1996; Schildkraut, 1965). One of the genetic disorders associated with this element is Wilson's disease, where among a wide range of symptoms, behavioural psychiatric-like abnormalities occur (Rathbun, 1996). In recent years it has been recognized that immunological alterations, such as acute phase proteins, accompany major depression (Maes, 1995). The acute phase, among other things, is characterized by increased hepatic synthesis of coeruloplasmin and metallothionein (Maes et al., 1992; Bremner and Beatie, 1990). These two proteins are connected with the biological role of copper. Copper is associated with at least ®ve major components of the blood plasma. Coeruloplasmin, *Correspondence to: M. Schlegel-Zawadzka, Department of Food Chemistry and Nutrition, Collegium Medicum, Jagiellonian University, Medyczna 9, 30-688 KrakoÂw, Poland. Tel: 48-12-658-3577, ext. 4850. Fax: 48-12-654-3949. E-mail:
[email protected]
CCC 0885±6222/99/070447±05$17.50 Copyright # 1999 John Wiley & Sons, Ltd.
recognized as containing the largest proportion of copper in plasma, albumin, two larger, noncoeruloplasmin nonalbumin proteins Ð ferroxidase II and transcuprein, and a fraction of the plasma comprising small molecules, among them histidine. Most of these components are present in plasma as well as in cerebrospinal ¯uid (Linder, 1991). There are not many studies examining copper and its role in central (animal and human brain) and peripheral (blood) pools during aective disorders and antidepressant treatment (Hansen et al., 1983; Maes et al., 1997; Manser et al., 1989; Narang et al., 1991; Schlegel-Zawadzka et al., 1998; Schlegel-Zawadzka and Nowak, in press). To explore the possibilities of interpreting copper alterations in depression, we decided to compare animal models of depression with the results from a human study. As animal models we have chosen: chronic stress (named here as ``chronic severe stress'', CSS), chronic mild stress (CMS) and olfactory bulbectomy (OB) in rats. MATERIALS AND METHODS All procedures were conducted according to NIH Animal Care and Use Committee guidelines, and
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approved by the Ethical Committee of the Institute of Pharmacology, KrakoÂw and Medical University School, Lublin. The clinical part was approved by the Ethical Committee of Collegium Medicum, Jagiellonian University, KrakoÂw. CSS procedure Male Wistar rats (approx. 200 g) were housed six to a cage with food and water freely available, and maintained on a 12 h light±dark cycle (lights on at 8.00 a.m.). One group of animals was subjected to a CSS procedure for a period of 16 consecutive days ( for details see Katz et al., 1981). The stress regime consisted of two periods of: electric footshock (3 mA/0.2 s/2 s 10, 20 s), immobilization (temp. 208C, 2 h), electric bell (5 min), immobilization (temp. 48C, 2 h), cold swim (temp. 128C, 3 min), light (80 klx, 5 min), and food deprivation (48 h). The second group (sham) of animals was housed in a separate room and had no contact with the stressed animals. CMS procedure Male Wistar rats (approx. 200 g) were housed singly in plastic cages (40 25 15 cm) with food and water freely available, and maintained on a 12 h light±dark cycle (lights on at 8.00 a.m.). One group of animals was subjected to a CMS procedure for a period of eight consecutive weeks ( for details see Willner et al., 1992). Each week of stress regime consisted of: two periods of food and water deprivation, two periods of 458 cage tilt, two periods of intermittent illumination (light on and o every 2 h), two periods of soiled cage (200 ml water in sawdust bedding), two periods of paired housing, two periods of low intensity stroboscopic illumination (150 ¯ashes/min), and two periods of no stress. All stressors were of a 12±14 h duration and were applied continuously, day and night. The second group (sham) of animals was housed in a separate room and had no contact with the stressed animals. OB procedure Male Wistar rats (approx. 200 g) were housed four to a cage with food and water freely available, and maintained on a 12 h light±dark cycle (lights on at 8.00 a.m.). Bilateral olfactory bulbectomy was performed in rats anaesthetized with Vetbutal1 Copyright # 1999 John Wiley & Sons, Ltd.
(Biowet, Puøawy, Poland) ( for details see Van Riezen and Leonard, 1991). Twenty-four hours after the last stressors or 5 weeks after OB surgery, the animals were decapitated, the trunk blood was collected and the serum separated, frozen and stored at ÿ208C for 1±2 months before assay. Clinical procedure Our clinical trial consisted of two groups: 19 major depressed patients (unipolar) and 16 control healthy volunteers. The severity of depression was measured by the Hamilton Depression Rating Scale (17 items; Hamilton, 1960). Blood was always drawn at 8.00 a.m., the serum separated, frozen and stored at ÿ208C for 1±2 months before assay. Copper assay procedure The measurement of the copper concentration in the serum was carried out as follows: the blank, 5 per cent glycerol aqueous solution; the composition of the diluted standards was: 1 : 5 (0.5 ml of standard 2.0 ml of 5 per cent aqueous glycerol solution). The samples studied and serum control were dissolved 1 : 5 in redistilled and deionized water. As serum control for the copper measurement Validate-N ser. B5-308 (Organon Teknika, USA) was used. The aqueous-glycerol standard for serum was Cu(NO3)2 from the Polish Committee for Standardisation, Measures and Analysis Control, Poland. The copper concentration in serum was determined using ¯ame atomic absorption spectrometry (Perkin Elmer 5100 PC equipped with a 5100 ZL Zeeman Furnace Module). The main instrumental parameters for the analysis by AAS were as follows: analytical wavelength 324.8 nm; slit width 0.7 nm; ¯ame acetylene ¯ow 2 l/min; air ¯ow 5 l/min; time of measurement for every sample 4 s; temperature 23008C. Group dierences were assessed using the unpaired t-test or Fisher's Exact Test. Relationships between variables were assessed using Spearman's rank order correlation coecient. Data were deemed signi®cant when p 5 0.05. RESULTS The means+SD of the age of the depressed and the control groups were 42.2+10.6 and 37.0+9.1 years, respectively, and were not signi®cantly dierent ( p 0.126, t-test). There were no Hum. Psychopharmacol. Clin. Exp. 14, 447±451 (1999)
SERUM COPPER IN ANIMAL AND HUMAN DEPRESSION
Table 1.
Serum copper concentration (mg/l) Control (n)
CSS CMS OB Clinical trial
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1.22+0.20 1.56+0.19 1.69+0.12 0.95+0.09
(11) (8) (6) (16)
Depression (n) 1.35+0.24 1.52+0.17 1.68+0.22 1.15+0.17
(11) (8) (10) (19)
Per cent of control 111 97 99 121*
Results are expressed as mean+SD of n subjects per group. CSS chronic severe stress; CMS chronic mild stress; OB olfactory bulbectomy. *p 5 0.05.
signi®cant dierences in the ratio male/female between depressed (7/12) and control groups (10/6) (Fisher's Exact Test). The mean+SD of the Hamilton Depression Rating Scale (HDRS) was 18.9+5.3 in the depressed patients. There was no signi®cant correlation between the serum copper and HDRS (r ÿ0.2241, p 0.3564). No dierences in the serum copper concentrations (mean+SD) was found between the sexes in the control groups (male 0.97+0.08 mg/l; female 0.93+0.10 mg/l). However, the serum copper concentrations (mean+SD) in the depressed groups diers between the sexes [male 1.05+ 0.15 mg/l; female 1.29+0.13 mg/l; t 2.795 (17), p 0.0124]. The serum copper concentration in the depressed group was signi®cantly higher (by 21 per cent) than in the control group [t 4.217 (33), p 0.0002, Table 1]. There was a signi®cant correlation between the age and the serum copper in the depressed group (r 0.5911, p 0.0077, Fig. 1a). This phenomenon did not exist in the control group (r 0.1074, p 0.6922, Fig. 1b). Three animal models of depression (CSS, CMS and OB) did not show any alterations in the serum copper concentration (Table 1). DISCUSSION Animal models of depression are used to study the mechanism(s) of pathophysiology (aetiology factors, pathogenesis) and treatment of that illness. Animal models demonstrate several behavioural and biochemical alterations, some of which are similar, some dierent to those found in human depression, and moreover, some changes present are speci®c for a particular model (Willner, 1990; Willner et al., 1992). Serum copper change seems to Copyright # 1999 John Wiley & Sons, Ltd.
Figure 1. The relationship between serum copper concentration and age of depression (a) and control (b) group.
be such an alteration which is not present in the animal models we examined, whilst it is demonstrated in human depression. Our previous study of brain and serum copper levels in rats after chronic antidepressant treatments showed dierences, depending on the treatment used, i.e. whether by drugs or electroconvulsive shock (SchlegelZawadzka et al., 1998; Schlegel-Zawadzka and Nowak, in press). Chronic treatment with either citalopram or imipramine decreased by 14.0 per cent and 13.5 per cent, respectively, the copper level in the serum, while electroconvulsive shock had no in¯uence. These two dierent types of antidepressant treatment had dierent eects on the brain copper content. Antidepressants had no eect, while electroconvulsive shock signi®cantly increased the copper level in the hippocampus (by 35.7 per cent) and the cerebellum (by 15.6 per cent). Our unpublished data indicate that copper (with a similar potency to zinc) inhibits ligand binding ([3H]MK-801) to the NMDA receptor-coupled ion Hum. Psychopharmacol. Clin. Exp. 14, 447±451 (1999)
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channel. It is to be noted that Doreulee et al. (1997), in an electrophysiological study, demonstrated the potency of copper to inhibit NMDAmediated activity in the hippocampus. These preliminary data implicate the copper ion as a potential modulator of this receptor complex, a complex which plays an important role in physiological and pathophysiological mechanisms in the central nervous system (see Nowak and SchlegelZawadzka, 1999). All these data show that the antidepressant-implicated mechanisms by which the copper concentration is altered have a complex nature and need further study. Several studies have been performed by an examination of serum copper concentrations in depressed humans. Narang et al. (1991) found in depressed patients an increased serum copper level (1.22 mg Cu/l compared with normal healthy individuals 1.07 mg Cu/l). The increase (14 per cent) was lower than in our studies (21 per cent, Table 1). Studies made of a Karachi population of psychiatric patients with depression gave similar results to ours Ð depressed patients had a higher (22 per cent) blood copper concentration (1.14 mg Cu/l compared with normal healthy individuals 0.94 mg Cu/l; Manser et al., 1989). In a European study, Maes et al. (1997) examined the blood of depressed patients for their serum copper concentration. Their results dier from ours. Maes et al. (1997) did not observe dierences between the serum copper concentrations of normal control and major depressed subjects. In their study, the serum copper was in¯uenced neither by age nor by sex of the subject. However, in their previous study they observed an increased serum concentration of coeruloplasmin (a major transport protein of copper) in depression (Maes et al., 1992; see Introduction). The discrepancy between Maes' data (Maes et al., 1997) and ours may be related to the dierent population of patients used [diagnosed as subgroups, treatment resistant depression (TRD) patients and non-treatment resistant depression (non-TRD) patients]. Indeed, non-TRD patients exhibit higher (by 24 per cent) serum copper levels (Maes et al., 1997). Moreover, the groups they studied were older than ours (control 47.5+15 years, major depression 51.4+13.5 years). It is known that the mean normal value of the plasma copper concentration increases with age (20±69 years) from 0.78 to 0.90 mg Cu/l for men and 1.01 to 1.14 mg Cu/l for women, respectively (Milne and Johnson, 1993). Copyright # 1999 John Wiley & Sons, Ltd.
The serum copper concentrations in our study in depressed patients was higher by about 21 per cent in comparison with the control group. In contrast, the zinc study undertaken by us in the same group of depressed patients showed about a 12 per cent decrease of the serum zinc concentration (Nowak et al., 1999). These results are in agreement with the well known fact of copper±zinc antagonistic interactions (Solomons, 1988). Increased serum copper and coeruloplasmin levels (and decreased serum zinc) are associated with activation of the in¯ammatory response system (IRS) and an acute phase (AP) response (Solomons, 1988). Indeed, IRS and AP has been demonstrated in human depression (Maes et al., 1997; Vandoolaeghe et al., 1999). Copper, besides its involvement in immune functions, possesses a signi®cant role in erythropoiesis (Solomons, 1988) which is also altered in human depression (Vandoolaeghe et al., 1999). The present study showed no relationship between the serum copper concentrations and severity of depression (HDRS), but there was a positive correlation with age in depressed patients indicating that the disorder is linked with increasing level of serum copper with age. This is clearly demonstrated in a younger ( present results), but not older (Maes et al., 1997), population of patients. The relevance of these ®ndings to the pathophysiology of depression needs further clinical study. ACKNOWLEDGEMENTS The authors thank Drs. G. Ossowska (Department of Pharmacology, Lublin) and M. Papp (Institute of Pharmacology, KrakoÂw) for their agreement to collect blood from animal models of depression and Professor S. F. A. Kettle (UEA, Norwich, UK) for helpful comments. REFERENCES Bremner, I. and Beatie, J. H. (1990). Metallothionein and the trace minerals. Annual Review of Nutrition, 10, 63±83. Doreulee, N., Yanovsky, Y. and Hassa, H. L. (1997). Suppression of long-term potentiation in hippocampal slices by copper. Hippocampus, 7, 666±669. Hamilton, M. (1960). A rating scale for depression. Journal of Neurology Neurosurgery and Psychiatry, 23, 56±61. Hansen, C. R., Malecha Jr., M., Mackenzie, T. B. and Kroll, J. (1983). Copper and zinc de®ciencies in Hum. Psychopharmacol. Clin. Exp. 14, 447±451 (1999)
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Hum. Psychopharmacol. Clin. Exp. 14, 447±451 (1999)
