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Lower serum zinc in major depression in relation to changes in serum acute phase proteins.
Journal of Affective Disorders 56 (1999) 189–194 www.elsevier.com / locate / jad
Brief report
Lower serum zinc in major depression in relation to changes in serum acute phase proteins. Michael Maes a
a,b,c ,
*, Nathalie De Vos a , Paul Demedts d , Annick Wauters d , Hugo Neels d
Clinical Research Center for Mental Health ( CRC-MH), University Department of Psychiatry, AZ stuivenberg, 267 Lange Beedekensstraat, 2060 Antwerp, Belgium b IRCCS, Istituto Fatebenefratteli, Brescia, Italy c Department of Psychiatry, Vanderbilt University, Nashville, TN, USA d Laboratories of Clinical Biology, OCMW Hospitals, Antwerp, Belgium Received 19 August 1998; received in revised form 6 November 1998; accepted 9 January 1999
Abstract There is now some evidence that major depression is accompanied by activation of the inflammatory response system (IRS). Other signs of IRS activation, which have been reported in major depression are lowered serum zinc (Zn) and serum albumin (Alb) concentrations. In serum, Zn is closely bound to Alb. The aims of the present study were to replicate previous findings that major depression is accompanied by lowered serum Zn and Alb and to examine whether the decrease in serum Zn may be explained by that in serum Alb. The above variables were determined in 48 major depressed patients and in 15 age–sex-matched healthy volunteers. Serum Zn and Alb were significantly lower in major depressed patients than in normal volunteers. In healthy volunteers and major depressed patients, there were significant and positive correlations between serum Zn and Alb. We found that 53.8% of the variance in serum Zn could be explained by the combined effects of serum Alb and diagnostic classification. The results suggest that lower serum Zn in depression is in part explained by lowered serum Alb and by another depression-related mechanism. It is suggested that lower serum Zn in depression may be secondary to sequestration of metallothionein in the liver, which may be related to increased production of interleukin-6. 1999 Elsevier Science B.V. All rights reserved. Keywords: Acute phase response; Albumin; Cytokines; Depression; Immunity; Zinc
1. Introduction There is now evidence that depression is accom-
panied by an activation of the inflammatory response system (IRS) (review: Maes, 1997). (1) Increased numbers of leukocytes, monocytes, neutrophils, acti-
*Corresponding author. Present address: Clinical Research Center for Mental Health, University Department of Psychiatry, AZ Stuivenberg, 267 Lange Beedekensstraat, 2060 Antwerp, Belgium. Fax: 1 32-3-2177752. E-mail address: [email protected] (M. Maes) 0165-0327 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0165-0327( 99 )00011-7
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M. Maes et al. / Journal of Affective Disorders 56 (1999) 189 – 194
vated T-lymphocytes, and increased secretion of neopterin and prostaglandins (Muller et al., 1993; Perini et al., 1995; Seidel et al., 1995; Sluzewska et al., 1996a; Maes, 1997; Bonaccorso et al., 1998; Song et al., 1998). (2) An acute phase (AP) response as indicated by changes in serum AP proteins (Joyce et al., 1992; Song et al., 1994; Seidel et al., 1995; Sluzewska et al., 1996b; Maes, 1997). (3) Increased in or ex vivo secretion of proinflammatory cytokines, such as IL-1b, IL-6 and interferon-g (IFNg) (Seidel et al., 1995, 1996; Sluzewska et al., 1996a; Frommberger et al., 1997; Maes, 1997; Song et al., 1998). Since these proinflammatory cytokines induce IRS activation, the above changes in depression may be caused by increased production of IL-1b, IL-6 and IFNg (Maes, 1997). IRS activation is also accompanied by a fall in serum zinc (Zn) (Solomons, 1988; Srinivas et al., 1988; Mussalo-Rauhamaa et al., 1988). Zn is, in the plasma, firmly bound to a 2 -macroglobulin (40%), while the remaining Zn is loosely bound to Alb (55%) or amino acids (5%). The loosely bound Zn fraction provides the Zn delivery to the tissues (Solomons, 1988). Lowered serum Zn during IRS activation may be secondary to sequestration of the intracellular heavy metal binding protein metallothionein in the liver, which, in turn, may be related to an increased production of the proinflammatory cytokines, IL-1 and IL-6 (Cousins and Leinart, 1988; Morimoto et al., 1989; Van Miert et al., 1990). There is now evidence that depression is accompanied by lower serum Zn (Little et al., 1989; McLoughlin and Hodge, 1990; Maes et al., 1994, 1997). Because IRS activation results in decreased serum Alb concentrations, there is potentially less Zn binding protein available, which could in part explain lower serum Zn (Giroux et al., 1977; Goldblum et al., 1987; Kushner, 1993). However, it is not known whether the decrease in serum Zn in depression is attributable to lower serum Alb. We found that subchronic treatment with antidepressants did not significantly alter serum Zn (Maes et al., 1997). The aims of the present study were to: (i) replicate previous findings that serum Zn is lowered in depression; (ii) examine whether the decrease in serum Zn can be explained by lowered serum Alb; and (iii) examine the effects of subchronic treatment with antidepressants on serum Zn.
2. Subjects and methods
2.1. Subjects Fifteen healthy volunteers and 48 major depressed patients participated. The latter were admitted to the University Department of Psychiatry, AZ Stuivenberg, Antwerp, Belgium, between October 1996 and January 1998. All subjects gave informed consent after the study design was fully explained. The study protocol was approved by the institutional review board. Patients were classified as having major depression according to DSM-III-R criteria (APA, 1987), using the structured interview of the DSM-IIIR (Spitzer et al., 1990). Severity of depression was measured by means of the 17-item Hamilton Depression Rating Scale, HDRS (Hamilton, 1960). Twelve major depressed patients showed melancholic features. Patients with other axis I diagnoses were excluded. We excluded patients who had been taking fluoxetine, MAOIs, antipsychotic drugs, anticonvulsants or lithium and who were treated with ECT during the index episode. Healthy volunteers were free of any medication for at least 1 month prior to blood sampling. No one had ever been taking psychotropic drugs or was a regular drinker. Normal volunteers were excluded for past, present and family history of psychiatric disorders. Exclusionary criteria for subjects are: (i) medical illnesses; (ii) infectious or allergic reactions for 2 weeks prior to the study; and (iii) drugs known to affect Zn metabolism, immune or endocrine functions. After admission into hospital, all psychotropic drugs were discontinued and, consequently, patients underwent a wash-out period of 10 days. In patients, blood was drawn 10 days after admission into hospital. Eighteen depressed patients had been taking antidepressant drugs and 22 patients had been taking benzodiazepines (di-K-chlorazepate or lorazepam) before the 10-day wash-out period. Sixteen depressed patients were treated with benzodiazepines during the study period.
2.2. Methods In healthy volunteers and major depressed subjects fasting blood (overnight) was collected at 07:45 h. Zn was determined by means of an atomic absorp-
M. Maes et al. / Journal of Affective Disorders 56 (1999) 189 – 194
tion spectrometric method (Perkin-Elmer 2380). The interassay coefficient of variation (CV) was 8.5% (n 5 25). Total serum protein (TSP) was determined by means of the Kodak Ektachem Analyzer (Kodak, Vilvoorde, Belgium). The interassay CV was 2.2%. The percentage of Alb was determined electrophoretically by means of the Beckman Paragon SPE agarose system (Analis, Belgium). The interassay CV value was 3.1% (n 5 26). Serum haptoglobin was assayed by means of laser nephelometry (Behringwerke AG Marburg / Lahn, Germany). The interassay CV value was 7.1%. Eighteen major depressed patients had blood samplings for assay of serum Zn both before therapy (baseline) and after treatment with antidepressants during a 5-week period (endpoint). Patients were treated with fluoxetine 20 mg / day (n 5 6); fluoxetine 20 mg / day 1 pindolol 7.5 mg / day (n 5 5); or fluoxetine 20 mg / day 1 mianserin 30 mg / day (n 5 7). The 17-item HDRS score was completed 10 days after admission into hospital (baseline) and 5 weeks later (endpoint). A good clinical response was defined as a 50% or greater decrease in the 17-item HDRS score from baseline to 5 weeks later. Accordingly, patients were divided into responders (n 5 10) and non-responders (n 5 8).
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differences in men / women ratio ( x 2 5 2.0, df 5 1, P 5 0.1) between depressed (13 / 35) and healthy (7 / 8) subjects. There were no significant relationships between serum Zn and either age (r 5 0.09, P 5 0.5) or gender (F 5 1.7, df 5 1 / 57, P 5 0.2). Fig. 1 shows that serum Zn was significantly lower in depressed than in healthy subjects (F 5 20.2, df 5 1 / 55, P 5 0.0001; results of ANCOVA with age and sex as covariates). In depression, there were no significant correlations between serum Zn and the HDRS (mean HDRS 5 22.364.9; r 5 0.05, P 5 0.8). There were no significant correlations between Zn and duration of illness (r 5 0.23, P 5 0.1). There were no significant differences in serum Zn between depressed patients with and without melancholic features (F 5 0.3 df 5 1 / 40, P 5 0.6). Multiple regression analysis did not show any significant effects (F 5 1.4, df 5 3 / 40, P 5 0.2) of use of antidepressants and benzodiazepines before the 10-day wash-out period and use of benzodiazepines during the study period. Serum Zn was significantly lower in the depressed patients who were free of any psychotropic drugs for at least 5 weeks and normal volunteers (F 5 9.8, df 5 1 / 26, P 5 0.004). There
2.3. Statistics Relationships between variables were checked by means of Pearson’s product moment, Spearman’s rank order correlation coefficients or multiple regression analysis. The independence of classification systems was assessed by means of analysis of contingence tables ( x 2 -test). Group mean differences were examined by means of analysis of variance (ANOVA) or covariance (ANCOVA). Repeated measure (RM) design ANOVA was employed in order to check the differences in baseline and endpoint serum Zn concentrations.
3. Results There were no significant differences (F 5 0.09, df 5 1 / 61, P 5 0.8) in age between depressed (mean 5 54.0614.1 years) and normal (mean 5 55.3613.0 years) subjects. There were no significant
Fig. 1. Scatterplot showing serum zinc in healthy volunteers (HV) and in major depressed patients (MD).
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were no significant relationships between serum Zn and the HDRS items weight loss or anorexia. TSP (68.566.5 vs. 73.364.9 mg / dl; F 5 7.5, df 5 1 / 54, P 5 0.008) and serum Alb (40.964.9 vs. 44.563.1 mg / dl; F 5 7.2, df 5 1 / 54, P 5 0.009) were significantly lower in depressed than in normal subjects. In normal controls (r 5 0.66, P 5 0.02) as well as in major depressed patients (r 5 0.44, P 5 0.003), there were significant and positive correlations between Zn and TSP. In normal controls (r 5 0.82, P 5 0.001) as well as in depressed patients (r 5 0.35, P 5 0.02) there were significant and positive correlations between serum Zn and serum Alb. Serum Alb and diagnostic classification (F 5 32.3, P 5 0.00002) explained 53.8% of the variance in serum Zn (F 5 30.3, df 5 2 / 52, P , 10 24 ). Fig. 2. shows that there was a significant relationship (r 5 0.41, P 5 0.01) between serum Zn and haptoglobin (F 5 3.5, df 5 2 / 35, P 5 0.04; coefficient 1, F 5 6.5, P 5 0.01; coefficient 2, F 5 5.7, P 5 0.02; mean 5 155.8669.7 mg / dl). Spearman’s correlation analysis, performed on the subjects with serum haptoglobin values . 150 mg / dl, showed a significant inverse correlation between serum Zn and Hp (r 5 2 0.52, P 5 0.017). In the 18 depressed patients who had repeated measurements of serum Zn, there were significant effects of time on the HDRS score (F 5 37.0, df 5 1 /
Fig. 2. Regression of serum zinc on serum haptoglobin (Hp, in square-root transformation).
16, P , 10 24 ; baseline HDRS 23.365.0 vs. endpoint HDRS 14.369.6) and a significant time X responder status interaction (F 5 35.9, df 5 1 / 16, P , 10 24 ). RM ANOVA showed no significant effect of time (F 5 1.1, df 5 1 / 16, P 5 0.3) on serum Zn and no significant time X responder status interaction (F 5 0.1, df 5 1 / 16, P 5 0.7). There were no significant differences in baseline or endpoint serum Zn between therapy responders (n 5 10) and nonresponders (n 5 8). There were significant differences in serum Zn between major depressed patients who had been treated for 5 weeks with antidepressants and the normal volunteers (F 5 20.4, df 5 1 / 29, P 5 0.0002).
4. Discussion This study replicates previous findings that major depressed subjects show significantly lowered serum Zn (Little et al., 1989; McLoughlin and Hodge, 1990; Maes et al., 1994, 1997). The major finding of this study is that there were significant positive relationships between serum Zn and serum Alb and, thus, TSP. Our findings on lowered serum Alb and, consequently, TSP in depression are in agreement with previous reports (Van Hunsel et al., 1996; Maes, 1997). Because Alb is the major Zn binding protein, less Zn binding protein is available and, thus, lower serum Zn could be the consequence of lower serum Alb (Goldblum et al., 1987). However, we found that serum Alb and diagnostic classification independently from each other explained an important part of the variance in serum Zn, suggesting that lower serum Zn is in part related to lowered Alb and that another depression-related mechanism is involved. There are several possible causes for decreased serum Zn and Alb concentrations in depression. (1) Decreased serum Zn and Alb are related to the anorexia and subsequent malnutrition. This hypothesis is less likely since we were unable to find significant relationships between serum Zn and anorexia or weight loss and since no nutritional deficiencies, either of the marasmic or kwashiorkorlike type, can be detected in depression (Maes, 1997; Maes et al., 1997). Reductions in serum Alb may not be assumed to reflect primary malnutrition when there is evidence for an AP response (Fleck, 1989).
M. Maes et al. / Journal of Affective Disorders 56 (1999) 189 – 194
(2) Lower serum Zn could be the consequence of hypothalamic–pituitary–adrenal (HPA) hyperfunction, which frequently occurs in depression (Maes et al., 1993). We were unable to find any significant relationships between serum Zn and basal or postdexamethasone cortisol values in depression (Maes et al., 1994).(3) The most plausible hypothesis is that lower serum Zn and Alb are related to IRS activation. Previously, we found significant inverse relationships between lower serum Zn and markers of IRS activation in depression, e.g. increased serum IL-6 (Maes et al., 1994, 1997). Here we report a significant inverse relationship between serum Zn and haptoglobin values . 150 mg / dl. The relationships between serum Zn and IL-6 or positive AP proteins in depression, strongly suggests that lower serum Zn is caused by increased production of IL-6 (Maes, 1997). Another finding of this study is that subchronic treatment with ‘serotonergic’ antidepressant therapies (Maes et al., 1998) did not significantly alter serum Zn levels. These results are in agreement with those of our previous report (Maes et al., 1997). In both above studies, post-treatment serum Zn in depressed patients was significantly lower than that in normal volunteers and no significant relationship between serum Zn and clinical responsivity to serotonergic antidepressant therapies could be found.
Acknowledgements The research reported was supported in part by the Funds for Scientific Research, Vlaanderen (FWO), the Clinical Research Center for Mental Health (CRC-MH), Antwerp; Belgium; and the Staglin Investigator Award (NARSAD, USA) to Dr M. Maes. The secretarial assistance of Mrs M. Maes is greatly appreciated.
References APA, 1987. Diagnostic and Statistical Manual of Mental Disorders, American Psychiatric Association, Washington DC. Bonaccorso, S., Lin, A., Verkerk, R., VanHunsel, F., Libbrecht, I., ´ S., DeClerck, L., Stevens, W., Biondi, M., Janca, A., Scharpe, Maes, M., 1998. Immune markers in fibromyalgia: comparison
193
with major depressed patients and normal volunteers. J. Affect. Disord. 48, 75–82. Cousins, R.J., Leinart, A.S., 1988. Tissue-specific regulation of zinc metabolism and metallothionein genes by interleukin-1. FASEB J. 2, 2884–2890. Fleck, A., 1989. Clinical and nutritional aspects of changes in acute-phase proteins during inflammation. Proc. Nutr. Soc. 48, 347–354. Frommberger, U.H., Bauer, J., Haselbauer, P., Fraulin, A., Riemann, D., Berger, M., 1997. Interleukin-6-(IL-6) plasma levels in depression and schizophrenia: comparison between the acute state and after remission. Eur. Arch. Psychiatry Clin. Neurosci. 247, 228–233. Giroux, E., Schechter, P.J., Schoun, J., Sjoerdsma, A., 1977. Reduced binding of added zinc in serum of patients with decompensated hepatic cirrhosis. Eur. J. Clin. Invest. 7, 71–73. Goldblum, S.E., Cohen, D.A., Jay, M., McClain, C.J., 1987. Interleukin 1-induced depression of iron and zinc: role of granulocytes and lactoferrin. Am. J. Physiol. 252, E27–E32. Hamilton, M., 1960. A rating scale for depression. J. Neurol. Neurosurg. Psychiatry 23, 56–61. Joyce, P.R., Hawes, C.R., Mulder, R.T., Sellman, J.D., Wilson, D.A., Boswell, D.R., 1992. Elevated levels of acute phase plasma proteins in major depression. Biol. Psychiatry 32, 1035–1041. Kushner, I., 1993. Regulation of the acute phase response by cytokines. Perspect. Biol. Med. 36, 611–622. Little, K.Y., Castellanos, X., Humphries, L.L., Austin, J., 1989. Altered zinc metabolism in mood disorder patients. Biol. Psychiatry 26, 646–648. Maes, M., 1997. The immune pathophysiology of major depression. In: Honig, A., van Praag, H.M. (Eds.), Depression: Neurobiological, Psychopathological and Therapeutic Advances, John Wiley, Chichester, pp. 197–215. Maes, M., Bosmans, E., Meltzer, H.Y., Scharpe, S., Suy, E., 1993. Interleukin-1b: a putative mediator of HPA-axis hyperactivity in major depression? Am. J. Psychiatry 150, 1189–1193. Maes, M., Scharpe, S., D’Haese, W., De Broe, M., Cosyns, P., 1994. Hypozincaemia in depression. J. Affect. Disord. 31, 135–140. Maes, M., Vandoolaeghe, E., Neels, H., Demedts, P., Wauters, A., Meltzer, H.Y., Altamura, C., Desnyder, R., 1997. Lower serum zinc in major depression is a sensitive marker of treatment resistance and of the immune / inflammatory response in that illness. Biol. Psychiatry 42, 349–358. Maes, M., Van Hunsel, F., Libbrecht, I., Campens, D., Meltzer, H.Y., 1998. Pindolol and mianserin augment the effects of fluoxetine in hospitalized major depressed patients, even those with treatment resistance. J. Clin. Psychopharmacol. (in press). McLoughlin, I.J., Hodge, J.S., 1990. Zinc in depressive disorder. Acta Psychiatr. Scand. 82, 451–453. Morimoto, A., Sakata, Y., Watanabe, T., Murakami, N., 1989. Characteristics of fever and acute-phase responses induced in rabbits by IL-1 and TNF. Am. J. Physiol. 256, 35–41. Muller, N., Hofschuster, E., Ackenheil, M., Mempel, W., Eckstein, R., 1993. Investigations of the cellular immunity during depression and the free interval: evidence for an immune
194
M. Maes et al. / Journal of Affective Disorders 56 (1999) 189 – 194
activation in affective psychosis. Progr. Neuropsychopharmacol. Biol. Psychiatry 17, 713–730. Mussalo-Rauhamaa, H., Konttinen, Y.T., Lehto, J., Honkanen, V., 1988. Predictive clinical and laboratory parameters for serum zinc and copper in rheumatoid arthritis. Ann. Rheum. Disord. 47, 816–819. Perini, G.I., Zara, M., Carraro, C., Tosin, C., Gava, F., Santucci, M.G., Valverde, S., Defranchis, G., 1995. Psychoimmunoendocrine effects of panic disorder. Hum. Psychopharmacol. 10, 461–465. Seidel, A., Arolt, V., Hunstiger, M., Rink, L., Behnisch, A., Kirchner, H., 1995. Cytokine production and serum proteins in depression. Scand. J. Immunol. 41, 534–538. Seidel, A., Arolt, V., Hunstiger, M., Rink, L., Behnisch, A., Kirchner, H., 1996. Increased CD56 1 natural killer cells and related cytokines in major depression. Clin. Immunol. Immunopathol. 78, 83–85. Sluzewska, A., Rybakowski, J., Bosmans, E., Sobieska, M., Berghmans, R., Maes, M., Wiktorowicz, K., 1996a. Indicators of immune activation in major depression. Psychiatry Res. 64, 161–167. Sluzewska, A., Rybakowski, J.K., Sobieska, M., Wiktorowicz, K., 1996b. Concentration and microheterogeneity glycophorms of alpha-1-acid glycoprotein in major depressive disorder. J. Affect. Disord. 39, 149–155. Solomons, N.W., 1988. Zinc and copper. In: Shils, M.E., Young,
V.R. (Eds.), Modern Nutrition in Health and Disease, Lea & Febiger, Philadelphia, pp. 238–262. Song, C., Dinan, T., Leonard, B.E., 1994. Changes in immunoglobulin, complement and acute phase protein levels in the depressed patients and normal controls. J. Affect. Disord. 30, 283–288. Song, C., Lin, A., Bonaccorso, S., Heide, C., Verkerk, R., Kenis, G., Bosmans, E., Scharpe, S., Cosyns, P., DeJong, R., Maes, M., 1998. The inflammatory response system and the availability of tryptophan to the brain of patients with major depression and sleep disorders. J. Affect. Disord. 49, 211–219. Spitzer, R.L., Williams, J.B.W., Gibbon, M., First, M.B., 1990. Structured Clinical Interview according to DSM-III-R, American Press, New York, NY. Srinivas, U., Braconier, J.H., Jeppsson, B., Abdulla, M., Akesson, B., Ockerman, P.A., 1988. Trace element alterations in infectious diseases. Scand. J. Clin. Lab. Invest. 48, 495–500. Van Hunsel, F., Wauters, A., Vandoolaeghe, E., Neels, H., Demedts, P., Maes, M., 1996. Lower total serum protein, albumin, and beta- and gamma-globulin in major and treatment-resistant depression: effects of antidepressant treatments. Psychiatry Res. 65, 159–169. Van Miert, A.S., Van Duin, C.T., Wensing, T., 1990. Fever and changes in plasma zinc and iron concentrations in the goat. The effects of interferon inducers and recombinant IFN-alpha 2a. J. Comp. Pathol. 103, 289–300.
Brief report
Lower serum zinc in major depression in relation to changes in serum acute phase proteins. Michael Maes a
a,b,c ,
*, Nathalie De Vos a , Paul Demedts d , Annick Wauters d , Hugo Neels d
Clinical Research Center for Mental Health ( CRC-MH), University Department of Psychiatry, AZ stuivenberg, 267 Lange Beedekensstraat, 2060 Antwerp, Belgium b IRCCS, Istituto Fatebenefratteli, Brescia, Italy c Department of Psychiatry, Vanderbilt University, Nashville, TN, USA d Laboratories of Clinical Biology, OCMW Hospitals, Antwerp, Belgium Received 19 August 1998; received in revised form 6 November 1998; accepted 9 January 1999
Abstract There is now some evidence that major depression is accompanied by activation of the inflammatory response system (IRS). Other signs of IRS activation, which have been reported in major depression are lowered serum zinc (Zn) and serum albumin (Alb) concentrations. In serum, Zn is closely bound to Alb. The aims of the present study were to replicate previous findings that major depression is accompanied by lowered serum Zn and Alb and to examine whether the decrease in serum Zn may be explained by that in serum Alb. The above variables were determined in 48 major depressed patients and in 15 age–sex-matched healthy volunteers. Serum Zn and Alb were significantly lower in major depressed patients than in normal volunteers. In healthy volunteers and major depressed patients, there were significant and positive correlations between serum Zn and Alb. We found that 53.8% of the variance in serum Zn could be explained by the combined effects of serum Alb and diagnostic classification. The results suggest that lower serum Zn in depression is in part explained by lowered serum Alb and by another depression-related mechanism. It is suggested that lower serum Zn in depression may be secondary to sequestration of metallothionein in the liver, which may be related to increased production of interleukin-6. 1999 Elsevier Science B.V. All rights reserved. Keywords: Acute phase response; Albumin; Cytokines; Depression; Immunity; Zinc
1. Introduction There is now evidence that depression is accom-
panied by an activation of the inflammatory response system (IRS) (review: Maes, 1997). (1) Increased numbers of leukocytes, monocytes, neutrophils, acti-
*Corresponding author. Present address: Clinical Research Center for Mental Health, University Department of Psychiatry, AZ Stuivenberg, 267 Lange Beedekensstraat, 2060 Antwerp, Belgium. Fax: 1 32-3-2177752. E-mail address: [email protected] (M. Maes) 0165-0327 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0165-0327( 99 )00011-7
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M. Maes et al. / Journal of Affective Disorders 56 (1999) 189 – 194
vated T-lymphocytes, and increased secretion of neopterin and prostaglandins (Muller et al., 1993; Perini et al., 1995; Seidel et al., 1995; Sluzewska et al., 1996a; Maes, 1997; Bonaccorso et al., 1998; Song et al., 1998). (2) An acute phase (AP) response as indicated by changes in serum AP proteins (Joyce et al., 1992; Song et al., 1994; Seidel et al., 1995; Sluzewska et al., 1996b; Maes, 1997). (3) Increased in or ex vivo secretion of proinflammatory cytokines, such as IL-1b, IL-6 and interferon-g (IFNg) (Seidel et al., 1995, 1996; Sluzewska et al., 1996a; Frommberger et al., 1997; Maes, 1997; Song et al., 1998). Since these proinflammatory cytokines induce IRS activation, the above changes in depression may be caused by increased production of IL-1b, IL-6 and IFNg (Maes, 1997). IRS activation is also accompanied by a fall in serum zinc (Zn) (Solomons, 1988; Srinivas et al., 1988; Mussalo-Rauhamaa et al., 1988). Zn is, in the plasma, firmly bound to a 2 -macroglobulin (40%), while the remaining Zn is loosely bound to Alb (55%) or amino acids (5%). The loosely bound Zn fraction provides the Zn delivery to the tissues (Solomons, 1988). Lowered serum Zn during IRS activation may be secondary to sequestration of the intracellular heavy metal binding protein metallothionein in the liver, which, in turn, may be related to an increased production of the proinflammatory cytokines, IL-1 and IL-6 (Cousins and Leinart, 1988; Morimoto et al., 1989; Van Miert et al., 1990). There is now evidence that depression is accompanied by lower serum Zn (Little et al., 1989; McLoughlin and Hodge, 1990; Maes et al., 1994, 1997). Because IRS activation results in decreased serum Alb concentrations, there is potentially less Zn binding protein available, which could in part explain lower serum Zn (Giroux et al., 1977; Goldblum et al., 1987; Kushner, 1993). However, it is not known whether the decrease in serum Zn in depression is attributable to lower serum Alb. We found that subchronic treatment with antidepressants did not significantly alter serum Zn (Maes et al., 1997). The aims of the present study were to: (i) replicate previous findings that serum Zn is lowered in depression; (ii) examine whether the decrease in serum Zn can be explained by lowered serum Alb; and (iii) examine the effects of subchronic treatment with antidepressants on serum Zn.
2. Subjects and methods
2.1. Subjects Fifteen healthy volunteers and 48 major depressed patients participated. The latter were admitted to the University Department of Psychiatry, AZ Stuivenberg, Antwerp, Belgium, between October 1996 and January 1998. All subjects gave informed consent after the study design was fully explained. The study protocol was approved by the institutional review board. Patients were classified as having major depression according to DSM-III-R criteria (APA, 1987), using the structured interview of the DSM-IIIR (Spitzer et al., 1990). Severity of depression was measured by means of the 17-item Hamilton Depression Rating Scale, HDRS (Hamilton, 1960). Twelve major depressed patients showed melancholic features. Patients with other axis I diagnoses were excluded. We excluded patients who had been taking fluoxetine, MAOIs, antipsychotic drugs, anticonvulsants or lithium and who were treated with ECT during the index episode. Healthy volunteers were free of any medication for at least 1 month prior to blood sampling. No one had ever been taking psychotropic drugs or was a regular drinker. Normal volunteers were excluded for past, present and family history of psychiatric disorders. Exclusionary criteria for subjects are: (i) medical illnesses; (ii) infectious or allergic reactions for 2 weeks prior to the study; and (iii) drugs known to affect Zn metabolism, immune or endocrine functions. After admission into hospital, all psychotropic drugs were discontinued and, consequently, patients underwent a wash-out period of 10 days. In patients, blood was drawn 10 days after admission into hospital. Eighteen depressed patients had been taking antidepressant drugs and 22 patients had been taking benzodiazepines (di-K-chlorazepate or lorazepam) before the 10-day wash-out period. Sixteen depressed patients were treated with benzodiazepines during the study period.
2.2. Methods In healthy volunteers and major depressed subjects fasting blood (overnight) was collected at 07:45 h. Zn was determined by means of an atomic absorp-
M. Maes et al. / Journal of Affective Disorders 56 (1999) 189 – 194
tion spectrometric method (Perkin-Elmer 2380). The interassay coefficient of variation (CV) was 8.5% (n 5 25). Total serum protein (TSP) was determined by means of the Kodak Ektachem Analyzer (Kodak, Vilvoorde, Belgium). The interassay CV was 2.2%. The percentage of Alb was determined electrophoretically by means of the Beckman Paragon SPE agarose system (Analis, Belgium). The interassay CV value was 3.1% (n 5 26). Serum haptoglobin was assayed by means of laser nephelometry (Behringwerke AG Marburg / Lahn, Germany). The interassay CV value was 7.1%. Eighteen major depressed patients had blood samplings for assay of serum Zn both before therapy (baseline) and after treatment with antidepressants during a 5-week period (endpoint). Patients were treated with fluoxetine 20 mg / day (n 5 6); fluoxetine 20 mg / day 1 pindolol 7.5 mg / day (n 5 5); or fluoxetine 20 mg / day 1 mianserin 30 mg / day (n 5 7). The 17-item HDRS score was completed 10 days after admission into hospital (baseline) and 5 weeks later (endpoint). A good clinical response was defined as a 50% or greater decrease in the 17-item HDRS score from baseline to 5 weeks later. Accordingly, patients were divided into responders (n 5 10) and non-responders (n 5 8).
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differences in men / women ratio ( x 2 5 2.0, df 5 1, P 5 0.1) between depressed (13 / 35) and healthy (7 / 8) subjects. There were no significant relationships between serum Zn and either age (r 5 0.09, P 5 0.5) or gender (F 5 1.7, df 5 1 / 57, P 5 0.2). Fig. 1 shows that serum Zn was significantly lower in depressed than in healthy subjects (F 5 20.2, df 5 1 / 55, P 5 0.0001; results of ANCOVA with age and sex as covariates). In depression, there were no significant correlations between serum Zn and the HDRS (mean HDRS 5 22.364.9; r 5 0.05, P 5 0.8). There were no significant correlations between Zn and duration of illness (r 5 0.23, P 5 0.1). There were no significant differences in serum Zn between depressed patients with and without melancholic features (F 5 0.3 df 5 1 / 40, P 5 0.6). Multiple regression analysis did not show any significant effects (F 5 1.4, df 5 3 / 40, P 5 0.2) of use of antidepressants and benzodiazepines before the 10-day wash-out period and use of benzodiazepines during the study period. Serum Zn was significantly lower in the depressed patients who were free of any psychotropic drugs for at least 5 weeks and normal volunteers (F 5 9.8, df 5 1 / 26, P 5 0.004). There
2.3. Statistics Relationships between variables were checked by means of Pearson’s product moment, Spearman’s rank order correlation coefficients or multiple regression analysis. The independence of classification systems was assessed by means of analysis of contingence tables ( x 2 -test). Group mean differences were examined by means of analysis of variance (ANOVA) or covariance (ANCOVA). Repeated measure (RM) design ANOVA was employed in order to check the differences in baseline and endpoint serum Zn concentrations.
3. Results There were no significant differences (F 5 0.09, df 5 1 / 61, P 5 0.8) in age between depressed (mean 5 54.0614.1 years) and normal (mean 5 55.3613.0 years) subjects. There were no significant
Fig. 1. Scatterplot showing serum zinc in healthy volunteers (HV) and in major depressed patients (MD).
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were no significant relationships between serum Zn and the HDRS items weight loss or anorexia. TSP (68.566.5 vs. 73.364.9 mg / dl; F 5 7.5, df 5 1 / 54, P 5 0.008) and serum Alb (40.964.9 vs. 44.563.1 mg / dl; F 5 7.2, df 5 1 / 54, P 5 0.009) were significantly lower in depressed than in normal subjects. In normal controls (r 5 0.66, P 5 0.02) as well as in major depressed patients (r 5 0.44, P 5 0.003), there were significant and positive correlations between Zn and TSP. In normal controls (r 5 0.82, P 5 0.001) as well as in depressed patients (r 5 0.35, P 5 0.02) there were significant and positive correlations between serum Zn and serum Alb. Serum Alb and diagnostic classification (F 5 32.3, P 5 0.00002) explained 53.8% of the variance in serum Zn (F 5 30.3, df 5 2 / 52, P , 10 24 ). Fig. 2. shows that there was a significant relationship (r 5 0.41, P 5 0.01) between serum Zn and haptoglobin (F 5 3.5, df 5 2 / 35, P 5 0.04; coefficient 1, F 5 6.5, P 5 0.01; coefficient 2, F 5 5.7, P 5 0.02; mean 5 155.8669.7 mg / dl). Spearman’s correlation analysis, performed on the subjects with serum haptoglobin values . 150 mg / dl, showed a significant inverse correlation between serum Zn and Hp (r 5 2 0.52, P 5 0.017). In the 18 depressed patients who had repeated measurements of serum Zn, there were significant effects of time on the HDRS score (F 5 37.0, df 5 1 /
Fig. 2. Regression of serum zinc on serum haptoglobin (Hp, in square-root transformation).
16, P , 10 24 ; baseline HDRS 23.365.0 vs. endpoint HDRS 14.369.6) and a significant time X responder status interaction (F 5 35.9, df 5 1 / 16, P , 10 24 ). RM ANOVA showed no significant effect of time (F 5 1.1, df 5 1 / 16, P 5 0.3) on serum Zn and no significant time X responder status interaction (F 5 0.1, df 5 1 / 16, P 5 0.7). There were no significant differences in baseline or endpoint serum Zn between therapy responders (n 5 10) and nonresponders (n 5 8). There were significant differences in serum Zn between major depressed patients who had been treated for 5 weeks with antidepressants and the normal volunteers (F 5 20.4, df 5 1 / 29, P 5 0.0002).
4. Discussion This study replicates previous findings that major depressed subjects show significantly lowered serum Zn (Little et al., 1989; McLoughlin and Hodge, 1990; Maes et al., 1994, 1997). The major finding of this study is that there were significant positive relationships between serum Zn and serum Alb and, thus, TSP. Our findings on lowered serum Alb and, consequently, TSP in depression are in agreement with previous reports (Van Hunsel et al., 1996; Maes, 1997). Because Alb is the major Zn binding protein, less Zn binding protein is available and, thus, lower serum Zn could be the consequence of lower serum Alb (Goldblum et al., 1987). However, we found that serum Alb and diagnostic classification independently from each other explained an important part of the variance in serum Zn, suggesting that lower serum Zn is in part related to lowered Alb and that another depression-related mechanism is involved. There are several possible causes for decreased serum Zn and Alb concentrations in depression. (1) Decreased serum Zn and Alb are related to the anorexia and subsequent malnutrition. This hypothesis is less likely since we were unable to find significant relationships between serum Zn and anorexia or weight loss and since no nutritional deficiencies, either of the marasmic or kwashiorkorlike type, can be detected in depression (Maes, 1997; Maes et al., 1997). Reductions in serum Alb may not be assumed to reflect primary malnutrition when there is evidence for an AP response (Fleck, 1989).
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(2) Lower serum Zn could be the consequence of hypothalamic–pituitary–adrenal (HPA) hyperfunction, which frequently occurs in depression (Maes et al., 1993). We were unable to find any significant relationships between serum Zn and basal or postdexamethasone cortisol values in depression (Maes et al., 1994).(3) The most plausible hypothesis is that lower serum Zn and Alb are related to IRS activation. Previously, we found significant inverse relationships between lower serum Zn and markers of IRS activation in depression, e.g. increased serum IL-6 (Maes et al., 1994, 1997). Here we report a significant inverse relationship between serum Zn and haptoglobin values . 150 mg / dl. The relationships between serum Zn and IL-6 or positive AP proteins in depression, strongly suggests that lower serum Zn is caused by increased production of IL-6 (Maes, 1997). Another finding of this study is that subchronic treatment with ‘serotonergic’ antidepressant therapies (Maes et al., 1998) did not significantly alter serum Zn levels. These results are in agreement with those of our previous report (Maes et al., 1997). In both above studies, post-treatment serum Zn in depressed patients was significantly lower than that in normal volunteers and no significant relationship between serum Zn and clinical responsivity to serotonergic antidepressant therapies could be found.
Acknowledgements The research reported was supported in part by the Funds for Scientific Research, Vlaanderen (FWO), the Clinical Research Center for Mental Health (CRC-MH), Antwerp; Belgium; and the Staglin Investigator Award (NARSAD, USA) to Dr M. Maes. The secretarial assistance of Mrs M. Maes is greatly appreciated.
References APA, 1987. Diagnostic and Statistical Manual of Mental Disorders, American Psychiatric Association, Washington DC. Bonaccorso, S., Lin, A., Verkerk, R., VanHunsel, F., Libbrecht, I., ´ S., DeClerck, L., Stevens, W., Biondi, M., Janca, A., Scharpe, Maes, M., 1998. Immune markers in fibromyalgia: comparison
193
with major depressed patients and normal volunteers. J. Affect. Disord. 48, 75–82. Cousins, R.J., Leinart, A.S., 1988. Tissue-specific regulation of zinc metabolism and metallothionein genes by interleukin-1. FASEB J. 2, 2884–2890. Fleck, A., 1989. Clinical and nutritional aspects of changes in acute-phase proteins during inflammation. Proc. Nutr. Soc. 48, 347–354. Frommberger, U.H., Bauer, J., Haselbauer, P., Fraulin, A., Riemann, D., Berger, M., 1997. Interleukin-6-(IL-6) plasma levels in depression and schizophrenia: comparison between the acute state and after remission. Eur. Arch. Psychiatry Clin. Neurosci. 247, 228–233. Giroux, E., Schechter, P.J., Schoun, J., Sjoerdsma, A., 1977. Reduced binding of added zinc in serum of patients with decompensated hepatic cirrhosis. Eur. J. Clin. Invest. 7, 71–73. Goldblum, S.E., Cohen, D.A., Jay, M., McClain, C.J., 1987. Interleukin 1-induced depression of iron and zinc: role of granulocytes and lactoferrin. Am. J. Physiol. 252, E27–E32. Hamilton, M., 1960. A rating scale for depression. J. Neurol. Neurosurg. Psychiatry 23, 56–61. Joyce, P.R., Hawes, C.R., Mulder, R.T., Sellman, J.D., Wilson, D.A., Boswell, D.R., 1992. Elevated levels of acute phase plasma proteins in major depression. Biol. Psychiatry 32, 1035–1041. Kushner, I., 1993. Regulation of the acute phase response by cytokines. Perspect. Biol. Med. 36, 611–622. Little, K.Y., Castellanos, X., Humphries, L.L., Austin, J., 1989. Altered zinc metabolism in mood disorder patients. Biol. Psychiatry 26, 646–648. Maes, M., 1997. The immune pathophysiology of major depression. In: Honig, A., van Praag, H.M. (Eds.), Depression: Neurobiological, Psychopathological and Therapeutic Advances, John Wiley, Chichester, pp. 197–215. Maes, M., Bosmans, E., Meltzer, H.Y., Scharpe, S., Suy, E., 1993. Interleukin-1b: a putative mediator of HPA-axis hyperactivity in major depression? Am. J. Psychiatry 150, 1189–1193. Maes, M., Scharpe, S., D’Haese, W., De Broe, M., Cosyns, P., 1994. Hypozincaemia in depression. J. Affect. Disord. 31, 135–140. Maes, M., Vandoolaeghe, E., Neels, H., Demedts, P., Wauters, A., Meltzer, H.Y., Altamura, C., Desnyder, R., 1997. Lower serum zinc in major depression is a sensitive marker of treatment resistance and of the immune / inflammatory response in that illness. Biol. Psychiatry 42, 349–358. Maes, M., Van Hunsel, F., Libbrecht, I., Campens, D., Meltzer, H.Y., 1998. Pindolol and mianserin augment the effects of fluoxetine in hospitalized major depressed patients, even those with treatment resistance. J. Clin. Psychopharmacol. (in press). McLoughlin, I.J., Hodge, J.S., 1990. Zinc in depressive disorder. Acta Psychiatr. Scand. 82, 451–453. Morimoto, A., Sakata, Y., Watanabe, T., Murakami, N., 1989. Characteristics of fever and acute-phase responses induced in rabbits by IL-1 and TNF. Am. J. Physiol. 256, 35–41. Muller, N., Hofschuster, E., Ackenheil, M., Mempel, W., Eckstein, R., 1993. Investigations of the cellular immunity during depression and the free interval: evidence for an immune
194
M. Maes et al. / Journal of Affective Disorders 56 (1999) 189 – 194
activation in affective psychosis. Progr. Neuropsychopharmacol. Biol. Psychiatry 17, 713–730. Mussalo-Rauhamaa, H., Konttinen, Y.T., Lehto, J., Honkanen, V., 1988. Predictive clinical and laboratory parameters for serum zinc and copper in rheumatoid arthritis. Ann. Rheum. Disord. 47, 816–819. Perini, G.I., Zara, M., Carraro, C., Tosin, C., Gava, F., Santucci, M.G., Valverde, S., Defranchis, G., 1995. Psychoimmunoendocrine effects of panic disorder. Hum. Psychopharmacol. 10, 461–465. Seidel, A., Arolt, V., Hunstiger, M., Rink, L., Behnisch, A., Kirchner, H., 1995. Cytokine production and serum proteins in depression. Scand. J. Immunol. 41, 534–538. Seidel, A., Arolt, V., Hunstiger, M., Rink, L., Behnisch, A., Kirchner, H., 1996. Increased CD56 1 natural killer cells and related cytokines in major depression. Clin. Immunol. Immunopathol. 78, 83–85. Sluzewska, A., Rybakowski, J., Bosmans, E., Sobieska, M., Berghmans, R., Maes, M., Wiktorowicz, K., 1996a. Indicators of immune activation in major depression. Psychiatry Res. 64, 161–167. Sluzewska, A., Rybakowski, J.K., Sobieska, M., Wiktorowicz, K., 1996b. Concentration and microheterogeneity glycophorms of alpha-1-acid glycoprotein in major depressive disorder. J. Affect. Disord. 39, 149–155. Solomons, N.W., 1988. Zinc and copper. In: Shils, M.E., Young,
V.R. (Eds.), Modern Nutrition in Health and Disease, Lea & Febiger, Philadelphia, pp. 238–262. Song, C., Dinan, T., Leonard, B.E., 1994. Changes in immunoglobulin, complement and acute phase protein levels in the depressed patients and normal controls. J. Affect. Disord. 30, 283–288. Song, C., Lin, A., Bonaccorso, S., Heide, C., Verkerk, R., Kenis, G., Bosmans, E., Scharpe, S., Cosyns, P., DeJong, R., Maes, M., 1998. The inflammatory response system and the availability of tryptophan to the brain of patients with major depression and sleep disorders. J. Affect. Disord. 49, 211–219. Spitzer, R.L., Williams, J.B.W., Gibbon, M., First, M.B., 1990. Structured Clinical Interview according to DSM-III-R, American Press, New York, NY. Srinivas, U., Braconier, J.H., Jeppsson, B., Abdulla, M., Akesson, B., Ockerman, P.A., 1988. Trace element alterations in infectious diseases. Scand. J. Clin. Lab. Invest. 48, 495–500. Van Hunsel, F., Wauters, A., Vandoolaeghe, E., Neels, H., Demedts, P., Maes, M., 1996. Lower total serum protein, albumin, and beta- and gamma-globulin in major and treatment-resistant depression: effects of antidepressant treatments. Psychiatry Res. 65, 159–169. Van Miert, A.S., Van Duin, C.T., Wensing, T., 1990. Fever and changes in plasma zinc and iron concentrations in the goat. The effects of interferon inducers and recombinant IFN-alpha 2a. J. Comp. Pathol. 103, 289–300.