Journal ofAffective Disorders, 22 (1991) 105-110 0 1991 Elsevier Science Publishers B.V. 0165-0327/Y ADONIS 016.503279100097)
, but it has not been found to be increased in mania (Lewis and McChesney, 1985; Meltzer and Arora, 1986; Muscettola et al., 1986), although Lewis and McChesney (1985) noted a trend to lower maxima1 imipramine binding (B,,,) values in those patients with a diagnosis of mania who displayed some symptoms of depression. This study aimed to explore further the platelet imipramine binding characteristics in manic patients and to compare the results with equivalent data for healthy controls and depressed patients.
Method Sixteen adult subjects admitted to a general hospital psychiatric unit and considered by their treating consultant to be suffering from mania were recruited into the study. None were suffering from a significant physical illness, organic mental disorder, or alcohol or drug dependence. Subjects were interviewed by a senior clinician (G.M.) to obtain demographic data, details of recent medication, a full account of previous psychiatric illnesses, any family history of psychiatric illness and to determine whether the patient met DSM-IIIR criteria for a manic episode (APA, 1987). The presence or absence of each DSM-IIIR symptom of a manic episode (criteria B, p. 217) and of a major depressive episode (criteria A, p, 222) was then recorded. Platelet imipramine binding in this group was compared to that in depressed patients (DSM-III major depression) and healthy control subjects examined in a concurrent study (Ellis et al., 1990). None of the patients suffering from mania reported here was included in the concurrent study. Imipramine binding assay Venous blood (40 ml) was collected into EDTA-containing vacutainers from an antecubital vein between 08.00 h and 11.00 h. Plateletrich plasma (PRP) was prepared by centrifugation at 100 X g max for 10 min at room temperature. The PRP was then stored at - 20 o C for up to 4 weeks. Platelet membranes were prepared by an adaptation of the technique of Barber et al. (1971) and the binding assay followed the method of Briley et al. (1979). Whole platelets were retrieved from PRP by centrifugation at 16,500 X g max for 10 min at room temperature and washed in buffer I (5 mM Tris, 20 mM EDTA, 150 mM NaCl, pH 7.5). The platelets were then preloaded with glycerol by spinning the cells through an isotonic O-40% glycerol gradient at 1465 X g max for 30 min at room temperature and then pelleting them at 5860 X g max for 10 min at room temperature. The pelleted cells were hypo-osmotically lysed by vortexing in buffer II (5 mM Tris, 5 mM EDTA, pH 7.5). Platelet membranes were then centrifuged at 39,000 X g max for 10 min at 4 o C and washed in buffer III (70 mM Tris, pH
7.5). The membranes were stored in assay buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, pH 7.4) at a concentration of 0.5-1.0 mg/ml and were kept at -20 o C for up to 1 week. Protein was assayed according to the method of Lowry et al. (19511, as modified by Markwell et al. (1978), using bovine serum albumin as the standard protein. “H-imipramine binding was measured at 4 o C using eight concentrations (1.0-8.0 nM) of the radioactive ligand (specific activity 20 Ci/mM, Amersham, U.K.). The incubation mixture contained a known amount of protein, close to 200 pg, in a total volume of 500 ~1. Specific binding was defined as the difference between total binding and that which remained in the presence of 50 PM desipramine. Following a l-h incubation, platelet membranes were washed rapidly with 16 ml cold assay buffer and filtered through Whatman GF/F glass fiber filters under vacuum. Filters were transferred to scintillation vials, airdried overnight, and extracted in a liquid scintillation cocktail before measurement for radioactivity. Estimates of the maximal receptor binding (B,,,) and the dissociation constant (K,) were obtained using an iterative, non-linear curve fitting technique (McIntosh and McIntosh, 1980) which has been shown to be superior to Scatchard plots (Scatchard, 1949; Cooke and McIntosh, 1986). The intra-assay coefficient of variation in binding was 6.3%, which was similar to that reported by others (Paul et al., 1980; Arora et al., 1986). Non-specific binding represented 30% of total binding at all concentrations of “H-imipramine employed. All samples were assayed with the laboratory blind to the clinical diagnosis. Comparisons between groups were made using non-parametric statistics in view of the skewed nature of some of the binding values. Results All subjects had suffered from a bipolar disorder for at least 1 year and 10 for more than 5 years. In the past, 13 subjects had suffered from both mania and major depression and the other three from major depression alone.
107
The principal finding was that maximal bindwas significantly higher (P < 0.05) in ing (B,,,) the manic group than in the depressed group and not significantly different to binding in the control group (see Table 1). As previously reported, binding was lower in the depressed group than in the control group. There was no significant difference in B,,, or K, between the sexes nor a significant correlation between age and binding parameters. There were no significant differences in age, sex or binding affinity between the different diagnostic groups. There was considerable homogeneity of symptoms within the manic group. Nearly every subject met all of the criteria for the category B items of DSM-IIIR mania. Some of the category A criteria for major depression were also satisfied. Nearly every subject suffered a change in sleep pattern and psychomotor agitation, and all but four reported a change in weight. Six patients had lost interest in their usual activities and seven noted a change in their appetite, but these two symptoms were not associated with any significant change in binding parameters. Six subjects exhibited a mixed mood state (although the predominant mood was elevated) but their imipramine binding levels were not decreased. There was no significant relationship between the total
TABLE
number of depressive symptoms experienced and B max values. The severity of mania was rated on an ordinal scale from 0 to 5, with 0 indicating no evidence of mania and 5, very severe mania. The mean rating for subjects was 3 (range 2-4; SD 0.7). When subjects were subdivided on the basis of these ratings, the binding parameters did not differ significantly among them (Kruskal-Wallis test). The duration of the current episode was classified as less than 2 weeks, or between 2 and 4 weeks, or longer than this. There were no significant differences in binding between these three subgroups (Kruskal-Wallis test), although the mean B,,, values for those who had been ill for more than 2 weeks (n = 9, mean B,,, f SD = 603 + 195 fmol/mg protein) was 25% lower than for those ill for a shorter period (n = 7, B,,, 803 k 239 fmol/mg protein) and the four subjects ill for more than 4 weeks at entry to the study had even lower numbers of binding sites (B,,, 499 f 159 fmol/mg protein). Three subjects had not taken any recent medication. Three had been treated with lithium, all for at least 3 months. Two had taken carbamazepine, one for 3 days and the other for 1 year. Twelve subjects had been treated with neuroleptics, nine for 3 days or less (of which six had been
I
COMPARISON OF CONTROL GROUPS
DEMOGRAPHIC
Group
n
DATA
Sex
AND
BINDING
VALUES
Age (years)
16 63 57 6 40
SM/8F 17M/46F 13M/44F 4M/2F 17M/23F
MANIC,
B,,, (fmol/mg
M/F
Mania a,b Major depression a Unipolar depression b Bipolar depression b Healthy control a
OF
DEPRESSED
protein)
AND
HEALTHY
K, (nM)
mean
SD
mean
SD
mean
SD
42.3 42.0 42.0 42.1 36.3
15.9 14.2 14.5 13.0 11.3
691 * 538 533 582 613 *
232 284 286 294 196
3.4 3.9 3.9 3.4 3.9
2.7 2.5 2.5 2.6 2.4
Data for depressed patients and healthy controls from Ellis et * Significantly higher than major depression group, P < 0.05, a Groups differ significantly, Kruskal-Wallis test H-corrected b Groups differ significantly, Kruskal-Wallis test H-corrected
al., 1990. Mann-Whitney U-test, for ties = 9.72, P = 0.02. for ties = 9.03 P = 0.01.
treated for 1 day only), two for a week and one for 4 weeks. The neuroleptics which had been prescribed were haloperidol (seven patients), thioridazine (three patients) and chlorpromazine (two patients). Discussion Imipramine binding among this group of patients suffering from mania did not differ significantly from the binding levels of healthy controls measured in a parallel study and were significantly higher than in depressed subjects. This is consistent with previous reports by others, but at some variance with a previous report (Ellis et al., 1990) that imipramine binding is reduced among a mixed group of non-depressed psychiatrically ill patients, which included some patients suffering from mania. However, while in that study an effort was made to examine patients after a washout period free of other medication, in this study patients were examined as soon as possible after presentation and therefore earlier in the course of their episode of mania. It is therefore of interest that the longer patients had been manic, the lower their B,,, values were and the mean B,,, values for the four subjects ill for 4 weeks or longer were somewhat lower than for the depressed group. As mania is a condition of rapid onset, in comparison to depression, it may be important to consider the presumptive mechanism by which changes in imipramine binding site numbers on platelets occur. The platelet is an anucleate cell and so unable to synthesize membrane proteins such as the imipramine binding site, which are presumptively produced at the megakaryocyte stage. Thus the expression of imipramine binding in the platelets represents a delayed phenomenon and it could be speculated that the higher levels of binding seen in those ill for only a short period may reflect their previous state of health (being comparable to control levels) while those later in the episode reflect the true level of binding associated with illness. A follow-up study would be necessary to explore this hypothesis further. Such a view also implies that the alteration in imipramine binding sites during affective disorder may be similar, whether the condition is mania or depression, which would
be consistent with the permissive hypothesis of serotonin function, which postulates that defective serotonergic dampening of other neurotransmitter systems permits the wide excursions seen in both depression and mania (Prange et al., 1974; Mendels and Frazer, 1975). However, the possible lack of specificity of imipramine binding as a biological marker for affective disorders must also be considered (Mellerup and Plenge, 1988; Ellis et al., 1990). It was not possible to confirm Lewis and McChesney’s (1985) finding of a trend to lower imipramine binding in those patients with some depressive symptoms associated with the episode of mania. Only 38% of subjects suffered from a mixed mood state, which is considerably lower than the figure of 72% quoted in a recent review of reports of mood symptoms in mania (Goodwin and Jamison, 19901, but this probably reflects that the subjects in this study were examined at an earlier stage of their illness. Although the values of B,,, in this study are lower than those described in some recent reports (Severson et al., 1990) they are similar to those found in the initial description of imipramine binding in human platelets (Briley et al., 1979) and in a number of more recent studies which employed a similar method for the determination of the amount of membrane protein in the assay (Langer et al., 1986; Roy et al., 1987; Maj et al., 1988; Takeda et al., 1989). It has been suggested that differences in the protein concentration in the final membrane preparation make a significant contribution to the variation in binding reported in different studies (Mellerup and Plenge, 1988) and this appears to be principally due to an effect on the binding affinity rather than the number of binding sites (Barkai et al., 1985). This may account for the higher K, values found in this study compared with those in which a lower final concentration of protein was employed (Mellerup et al., 1982). The time period during which samples were collected was longer than in some recent studies, which raises the possibility that circadian variation in binding may have been a confounding factor in this study. However, most studies in man have not found a consistent pattern of variation in binding during daylight hours (Gentsch et al.,
109
et al., 1989; Ellis et al., 19911, 1989; Montero although there may be a significant decrease at night (Nankai et al., 1986; Montero et al., 1989). Nearly all subjects were on continuing medication, or had recommenced medication prior to entry into the study, as the severity of their illness required treatment before the next 08.00-11.00 h sampling period. The possible effect of this medication on imipramine binding as a confounding factor in this study is an important consideration. However, binding is unaffected by treatment with lithium in healthy volunteers (Glue et al., 1986; Poirier et al., 1988) or by the discontinuation of lithium in euthymic bipolar patients (Goodnick et al., 1984) which suggests that treatment with lithium was unlikely to have altered binding values in this study. We are not aware of clinical studies of the effects of neuroleptic medication on imipramine binding, but in vitro studies of the pharmacological selectivity of different agents on imipramine binding in human platelets reported IC,,, values for amitriptyline, imipramine, desipramine and clomipramine in the range of 14-23 nM, while those for chlorpromazine were more than an order of magnitude greater at 300 nM (Langer et al., 1980) and for haloperidol more than two orders of magnitude greater at > 10,000 nM (Paul et al., 1980) with similar values described for trifluoperazine and thioridazine in rat brain (Rommelspacher and Strauss, 1985>, suggesting that these drugs have a low affinity for the imipramine binding site and are unlikely to have affected binding values in these patients. Further, in the parallel study using the same assay (Ellis et al., 1990). B,,, and K, values did not differ significantly between those non-depressed, psychiatrically ill patients free of psychotropic medication for 28 days and those medication-free for a shorter period or on continuing medication (predominantly neuroleptics), which supports the predictions from the in vitro studies. In the present study, only one subject had been treated with neuroleptics for more than a week and most patients had been taking them for only 1 or 2 days. It therefore appears unlikely that the observed decrease in binding with increasing duration of illness could be accounted for by differences in the duration of treatment with neuroleptics.
In conclusion, it is suggested that future studies of imipramine binding in mania should consider the duration of the illness and study the changes in binding throughout the episode of illness into recovery with a view to exploring similarities to imipramine binding in depressive illness. Acknowledgements This study was supported by the Medical Research Council of New Zealand. Desipramine was kindly donated by CIBA-Geigy New Zealand. References American Psychiatric Association (1987) Diagnostic and Statistical Manual of Mental Disorders, 3rd edn., revised. American Psychiatric Association, Washington, DC. Arora, R., Locascio, J.J. and Meltzer, H.Y. (1986) ‘H-imipramine binding in blood platelets of schizophrenic patients. Psychiatry Res. 19, 215-224. Barber, A.J.. Pepper, D.S. and Jamieson, G.A. (1971) A comparison of methods for platelet lysis and the isolation of platelet membranes. Thromb. Diath. Haemorrh. 26, 38-57. Barkai, A.I., Kowalik, S. and Baron, M. (1985) Effects of membrane protein concentration on binding of ‘H-imipramine in human platelets. Biol. Psychiatry 20, 215-219. Briley, MS.. Raisman, R. and Langer, S.Z. (1979) Human platelets possess high-affinity binding sites for ‘H-imipramine. Eur. J. Pharmacol. 58, 347-348. Cooke, R.C. and McIntosh, J.E.A. (1986) The analysis of data from breast cancer oestrogen and progesterone receptor assays: Scatchard plots are inferior to direct fitting by computer. Clin. Chim. Acta 154, 171&180. Ellis, P.M., McIntosh, C.J., Beeston, R.. Salmond, C.E., Cooke, R.R. and Mellsop, G. (1990) Platelet tritiated imipramine binding in psychiatric patients - relationship to symptoms and severity of depression. Acta Psychiatr. Stand. 82, 275-282. Ellis, P.M., Beeston, R., Cooke, R.R. and Mellsop, G. (1991) Circadian variation in platelet imipramine binding during the day in healthy subjects. Pharmacopsychiatry (in press). Gentsch, C., Lichtsteiner. M., Gastpar, M., Gastpar, G. and Feer, H. (1989) Platelet ‘H-imipramine binding sites in depressed patients and healthy controls: a comparison between morning and afternoon samples. J. Affect. Disord. 16, 65-70. Glue, P.W.. Cowen, P.J., Nutt, D.J., Kolakowska, T. and Grahame-Smith, D.G. (1986) The effect of lithium on 5-HT mediated neuroendocrine responses and platelet SHT receptors. Psychopharmacology 90, 398-402. Goodnick, P.J., Arora, R.C., Jackman, H. and Meltzer, H.Y. (1984) Neurochemical changes during discontinuation of
110
lithium prophylaxis. II: Alteration in platelet serotonin function. Biol. Psychiatry 19, 891-898. Goodwin, F.K. and Jamison, K.R. (1990) Manic-Depressive Illness. Oxford University Press, New York, p, 31. Langer, S.Z. and Schoemaker, H. (1988) Effects of antidepressants on monoamine transporters. Prog. Neuropsychopharmacol. Biol. Psychiatry 12, 193-216. Langer, S.Z., Briley, MS., Raisman, R., Henry, J.F. and Morselli, P.L. (19801 Specific ‘H-imipramine binding in human platelets: influence of age and sex. Naunyn-Schiedeberg’s Arch. Pharmacol. 313, 189-194. Langer, S.Z., Sechter, D., Loo, H., Raisman, R. and Zarifian, E. (1986) Electroconvulsive shock therapy and maximum binding of platelet tritiated imipramine binding in depression. Arch. Gen. Psychiatry 43, 949-952. Lewis, D.A. and McChesney, C. (19851 Tritiated imipramine binding to platelets in manic subjects. J. Affect. Disord. 9, 207-211. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randal, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Maj, M., Mastronardi, P., Cerreta, A., Romano, M., Mazzarella, B. and Kemali, D. (1988) Changes in platelet ‘H-imipramine binding in depressed patients receiving electroconvulsive therapy. Biol. Psychiatry 24, 469-472. Markwell, M.A.K., Hass, S.M., Bieber, L.L. and Tolbert, N.E. (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87, 206-210. McIntosh J.E.A. and McIntosh R.P. (19801 Mathematical Modelling and Computers in Endocrinology. Springer, Berlin. Mellerup, E.T. and Plenge, P. (1988) Imipramine binding in depression and other psychiatric conditions. Acta Psychiatr. Stand. 78 (Suppl. 3451, 61-68. Mellerup, E.T., Plenge, P. and Rosenberg, R. (1982) ‘H-imipramine binding sites in platelets from psychiatric patients. Psychiatry Res. 7, 221-227. Meltzer, H.Y. and Arora, R.C. (1986) Platelet markers of suicidality. Ann. NY Acad. Sci. 487. 271-280. Mendels, J. and Frazer, A. (1975) Reduced central serotonergic activity in mania: implications for the relationship between depression and mania. Br. J. Psychiatry 126, 241-248.
Montero, D., Ofori-Adjei, D. and Wagner, A. (19891 Circadian variation of platelet ‘H-imipramine binding, platelet serotonin content and plasma cortisol in healthy volunteers. Biol. Psychiatry 26, 7944804. Muscettola, G., Di Lauro, A. and Giannini, C.P. (19861 Platelet “H-imipramine binding in bipolar patients. Psychiatry Res. 18, 343-353. Nankai, M., Yoshimoto, S., Narita, K. and Takahashi, R. (19861 Platelet [“Hlimipramine binding in depressed patients and its circadian variation in healthy controls. J. Affect. Disord. 11, 207-212. Paul, S.M., Rehavi, M., Hulihan, B., Skolnick, P.\and Goodwin, F.K. (1980) A rapid and sensitive radioreceptor assay for tertiary amine tricyclic antidepressants. Commun. Psychopharmacol. 4, 487-494. Poirier, M.F., Galzin, A.M., Pimoule, C., Schoemaker, H., Le Quan Bui, K.H., Meyer, P., Gay, C., Loo, H. and Langer, S.Z. (19881 Short-term lithium administration to healthy volunteers produces long-lasting pronounced changes in platelet serotonin uptake but not imipramine binding. Psychopharmacol. 94, 521-526. Prange, A.J., Wilson, I.C., Lynn, C.W., Alltop, L.B. and Stikeleather, R.A. (19741 r.-Tryptophan in mania. Arch. Gen. Psychiatry 30, 56-62. Rommelspacher, H. and Strauss, S. (1985) Neuroleptics and p-carbolines displace (“Hl-imipramine for its binding sites in human and rat tissue. J. Neural Transmiss. 61, 55-63. Roy, A., Everett, D., Pickar, D. and Paul, S.M. (1987) Platelet tritiated imipramine binding and serotonin uptake in depressed patients and controls. Relationship to plasma cortisol levels before and after dexamethasone administration. Arch. Gen. Psychiatry 44, 320-327. Scatchard, G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660-672. Severson, J.A., Schneider, L.S. and Frederickson, E.R. (1990) Methodological issues in the preparation and assay of platelet ‘H-imipramine binding. Psychiatry Res. 33, 19-29. Takeda, T., Harada, T. and Otsuki, S. (1989) Platelet ‘Hclonidine and ‘HI-imipramine binding and plasma cortisol levels in depression. Biol. Psychiatry 26. 52-60.
, but it has not been found to be increased in mania (Lewis and McChesney, 1985; Meltzer and Arora, 1986; Muscettola et al., 1986), although Lewis and McChesney (1985) noted a trend to lower maxima1 imipramine binding (B,,,) values in those patients with a diagnosis of mania who displayed some symptoms of depression. This study aimed to explore further the platelet imipramine binding characteristics in manic patients and to compare the results with equivalent data for healthy controls and depressed patients.
Method Sixteen adult subjects admitted to a general hospital psychiatric unit and considered by their treating consultant to be suffering from mania were recruited into the study. None were suffering from a significant physical illness, organic mental disorder, or alcohol or drug dependence. Subjects were interviewed by a senior clinician (G.M.) to obtain demographic data, details of recent medication, a full account of previous psychiatric illnesses, any family history of psychiatric illness and to determine whether the patient met DSM-IIIR criteria for a manic episode (APA, 1987). The presence or absence of each DSM-IIIR symptom of a manic episode (criteria B, p. 217) and of a major depressive episode (criteria A, p, 222) was then recorded. Platelet imipramine binding in this group was compared to that in depressed patients (DSM-III major depression) and healthy control subjects examined in a concurrent study (Ellis et al., 1990). None of the patients suffering from mania reported here was included in the concurrent study. Imipramine binding assay Venous blood (40 ml) was collected into EDTA-containing vacutainers from an antecubital vein between 08.00 h and 11.00 h. Plateletrich plasma (PRP) was prepared by centrifugation at 100 X g max for 10 min at room temperature. The PRP was then stored at - 20 o C for up to 4 weeks. Platelet membranes were prepared by an adaptation of the technique of Barber et al. (1971) and the binding assay followed the method of Briley et al. (1979). Whole platelets were retrieved from PRP by centrifugation at 16,500 X g max for 10 min at room temperature and washed in buffer I (5 mM Tris, 20 mM EDTA, 150 mM NaCl, pH 7.5). The platelets were then preloaded with glycerol by spinning the cells through an isotonic O-40% glycerol gradient at 1465 X g max for 30 min at room temperature and then pelleting them at 5860 X g max for 10 min at room temperature. The pelleted cells were hypo-osmotically lysed by vortexing in buffer II (5 mM Tris, 5 mM EDTA, pH 7.5). Platelet membranes were then centrifuged at 39,000 X g max for 10 min at 4 o C and washed in buffer III (70 mM Tris, pH
7.5). The membranes were stored in assay buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, pH 7.4) at a concentration of 0.5-1.0 mg/ml and were kept at -20 o C for up to 1 week. Protein was assayed according to the method of Lowry et al. (19511, as modified by Markwell et al. (1978), using bovine serum albumin as the standard protein. “H-imipramine binding was measured at 4 o C using eight concentrations (1.0-8.0 nM) of the radioactive ligand (specific activity 20 Ci/mM, Amersham, U.K.). The incubation mixture contained a known amount of protein, close to 200 pg, in a total volume of 500 ~1. Specific binding was defined as the difference between total binding and that which remained in the presence of 50 PM desipramine. Following a l-h incubation, platelet membranes were washed rapidly with 16 ml cold assay buffer and filtered through Whatman GF/F glass fiber filters under vacuum. Filters were transferred to scintillation vials, airdried overnight, and extracted in a liquid scintillation cocktail before measurement for radioactivity. Estimates of the maximal receptor binding (B,,,) and the dissociation constant (K,) were obtained using an iterative, non-linear curve fitting technique (McIntosh and McIntosh, 1980) which has been shown to be superior to Scatchard plots (Scatchard, 1949; Cooke and McIntosh, 1986). The intra-assay coefficient of variation in binding was 6.3%, which was similar to that reported by others (Paul et al., 1980; Arora et al., 1986). Non-specific binding represented 30% of total binding at all concentrations of “H-imipramine employed. All samples were assayed with the laboratory blind to the clinical diagnosis. Comparisons between groups were made using non-parametric statistics in view of the skewed nature of some of the binding values. Results All subjects had suffered from a bipolar disorder for at least 1 year and 10 for more than 5 years. In the past, 13 subjects had suffered from both mania and major depression and the other three from major depression alone.
107
The principal finding was that maximal bindwas significantly higher (P < 0.05) in ing (B,,,) the manic group than in the depressed group and not significantly different to binding in the control group (see Table 1). As previously reported, binding was lower in the depressed group than in the control group. There was no significant difference in B,,, or K, between the sexes nor a significant correlation between age and binding parameters. There were no significant differences in age, sex or binding affinity between the different diagnostic groups. There was considerable homogeneity of symptoms within the manic group. Nearly every subject met all of the criteria for the category B items of DSM-IIIR mania. Some of the category A criteria for major depression were also satisfied. Nearly every subject suffered a change in sleep pattern and psychomotor agitation, and all but four reported a change in weight. Six patients had lost interest in their usual activities and seven noted a change in their appetite, but these two symptoms were not associated with any significant change in binding parameters. Six subjects exhibited a mixed mood state (although the predominant mood was elevated) but their imipramine binding levels were not decreased. There was no significant relationship between the total
TABLE
number of depressive symptoms experienced and B max values. The severity of mania was rated on an ordinal scale from 0 to 5, with 0 indicating no evidence of mania and 5, very severe mania. The mean rating for subjects was 3 (range 2-4; SD 0.7). When subjects were subdivided on the basis of these ratings, the binding parameters did not differ significantly among them (Kruskal-Wallis test). The duration of the current episode was classified as less than 2 weeks, or between 2 and 4 weeks, or longer than this. There were no significant differences in binding between these three subgroups (Kruskal-Wallis test), although the mean B,,, values for those who had been ill for more than 2 weeks (n = 9, mean B,,, f SD = 603 + 195 fmol/mg protein) was 25% lower than for those ill for a shorter period (n = 7, B,,, 803 k 239 fmol/mg protein) and the four subjects ill for more than 4 weeks at entry to the study had even lower numbers of binding sites (B,,, 499 f 159 fmol/mg protein). Three subjects had not taken any recent medication. Three had been treated with lithium, all for at least 3 months. Two had taken carbamazepine, one for 3 days and the other for 1 year. Twelve subjects had been treated with neuroleptics, nine for 3 days or less (of which six had been
I
COMPARISON OF CONTROL GROUPS
DEMOGRAPHIC
Group
n
DATA
Sex
AND
BINDING
VALUES
Age (years)
16 63 57 6 40
SM/8F 17M/46F 13M/44F 4M/2F 17M/23F
MANIC,
B,,, (fmol/mg
M/F
Mania a,b Major depression a Unipolar depression b Bipolar depression b Healthy control a
OF
DEPRESSED
protein)
AND
HEALTHY
K, (nM)
mean
SD
mean
SD
mean
SD
42.3 42.0 42.0 42.1 36.3
15.9 14.2 14.5 13.0 11.3
691 * 538 533 582 613 *
232 284 286 294 196
3.4 3.9 3.9 3.4 3.9
2.7 2.5 2.5 2.6 2.4
Data for depressed patients and healthy controls from Ellis et * Significantly higher than major depression group, P < 0.05, a Groups differ significantly, Kruskal-Wallis test H-corrected b Groups differ significantly, Kruskal-Wallis test H-corrected
al., 1990. Mann-Whitney U-test, for ties = 9.72, P = 0.02. for ties = 9.03 P = 0.01.
treated for 1 day only), two for a week and one for 4 weeks. The neuroleptics which had been prescribed were haloperidol (seven patients), thioridazine (three patients) and chlorpromazine (two patients). Discussion Imipramine binding among this group of patients suffering from mania did not differ significantly from the binding levels of healthy controls measured in a parallel study and were significantly higher than in depressed subjects. This is consistent with previous reports by others, but at some variance with a previous report (Ellis et al., 1990) that imipramine binding is reduced among a mixed group of non-depressed psychiatrically ill patients, which included some patients suffering from mania. However, while in that study an effort was made to examine patients after a washout period free of other medication, in this study patients were examined as soon as possible after presentation and therefore earlier in the course of their episode of mania. It is therefore of interest that the longer patients had been manic, the lower their B,,, values were and the mean B,,, values for the four subjects ill for 4 weeks or longer were somewhat lower than for the depressed group. As mania is a condition of rapid onset, in comparison to depression, it may be important to consider the presumptive mechanism by which changes in imipramine binding site numbers on platelets occur. The platelet is an anucleate cell and so unable to synthesize membrane proteins such as the imipramine binding site, which are presumptively produced at the megakaryocyte stage. Thus the expression of imipramine binding in the platelets represents a delayed phenomenon and it could be speculated that the higher levels of binding seen in those ill for only a short period may reflect their previous state of health (being comparable to control levels) while those later in the episode reflect the true level of binding associated with illness. A follow-up study would be necessary to explore this hypothesis further. Such a view also implies that the alteration in imipramine binding sites during affective disorder may be similar, whether the condition is mania or depression, which would
be consistent with the permissive hypothesis of serotonin function, which postulates that defective serotonergic dampening of other neurotransmitter systems permits the wide excursions seen in both depression and mania (Prange et al., 1974; Mendels and Frazer, 1975). However, the possible lack of specificity of imipramine binding as a biological marker for affective disorders must also be considered (Mellerup and Plenge, 1988; Ellis et al., 1990). It was not possible to confirm Lewis and McChesney’s (1985) finding of a trend to lower imipramine binding in those patients with some depressive symptoms associated with the episode of mania. Only 38% of subjects suffered from a mixed mood state, which is considerably lower than the figure of 72% quoted in a recent review of reports of mood symptoms in mania (Goodwin and Jamison, 19901, but this probably reflects that the subjects in this study were examined at an earlier stage of their illness. Although the values of B,,, in this study are lower than those described in some recent reports (Severson et al., 1990) they are similar to those found in the initial description of imipramine binding in human platelets (Briley et al., 1979) and in a number of more recent studies which employed a similar method for the determination of the amount of membrane protein in the assay (Langer et al., 1986; Roy et al., 1987; Maj et al., 1988; Takeda et al., 1989). It has been suggested that differences in the protein concentration in the final membrane preparation make a significant contribution to the variation in binding reported in different studies (Mellerup and Plenge, 1988) and this appears to be principally due to an effect on the binding affinity rather than the number of binding sites (Barkai et al., 1985). This may account for the higher K, values found in this study compared with those in which a lower final concentration of protein was employed (Mellerup et al., 1982). The time period during which samples were collected was longer than in some recent studies, which raises the possibility that circadian variation in binding may have been a confounding factor in this study. However, most studies in man have not found a consistent pattern of variation in binding during daylight hours (Gentsch et al.,
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et al., 1989; Ellis et al., 19911, 1989; Montero although there may be a significant decrease at night (Nankai et al., 1986; Montero et al., 1989). Nearly all subjects were on continuing medication, or had recommenced medication prior to entry into the study, as the severity of their illness required treatment before the next 08.00-11.00 h sampling period. The possible effect of this medication on imipramine binding as a confounding factor in this study is an important consideration. However, binding is unaffected by treatment with lithium in healthy volunteers (Glue et al., 1986; Poirier et al., 1988) or by the discontinuation of lithium in euthymic bipolar patients (Goodnick et al., 1984) which suggests that treatment with lithium was unlikely to have altered binding values in this study. We are not aware of clinical studies of the effects of neuroleptic medication on imipramine binding, but in vitro studies of the pharmacological selectivity of different agents on imipramine binding in human platelets reported IC,,, values for amitriptyline, imipramine, desipramine and clomipramine in the range of 14-23 nM, while those for chlorpromazine were more than an order of magnitude greater at 300 nM (Langer et al., 1980) and for haloperidol more than two orders of magnitude greater at > 10,000 nM (Paul et al., 1980) with similar values described for trifluoperazine and thioridazine in rat brain (Rommelspacher and Strauss, 1985>, suggesting that these drugs have a low affinity for the imipramine binding site and are unlikely to have affected binding values in these patients. Further, in the parallel study using the same assay (Ellis et al., 1990). B,,, and K, values did not differ significantly between those non-depressed, psychiatrically ill patients free of psychotropic medication for 28 days and those medication-free for a shorter period or on continuing medication (predominantly neuroleptics), which supports the predictions from the in vitro studies. In the present study, only one subject had been treated with neuroleptics for more than a week and most patients had been taking them for only 1 or 2 days. It therefore appears unlikely that the observed decrease in binding with increasing duration of illness could be accounted for by differences in the duration of treatment with neuroleptics.
In conclusion, it is suggested that future studies of imipramine binding in mania should consider the duration of the illness and study the changes in binding throughout the episode of illness into recovery with a view to exploring similarities to imipramine binding in depressive illness. Acknowledgements This study was supported by the Medical Research Council of New Zealand. Desipramine was kindly donated by CIBA-Geigy New Zealand. References American Psychiatric Association (1987) Diagnostic and Statistical Manual of Mental Disorders, 3rd edn., revised. American Psychiatric Association, Washington, DC. Arora, R., Locascio, J.J. and Meltzer, H.Y. (1986) ‘H-imipramine binding in blood platelets of schizophrenic patients. Psychiatry Res. 19, 215-224. Barber, A.J.. Pepper, D.S. and Jamieson, G.A. (1971) A comparison of methods for platelet lysis and the isolation of platelet membranes. Thromb. Diath. Haemorrh. 26, 38-57. Barkai, A.I., Kowalik, S. and Baron, M. (1985) Effects of membrane protein concentration on binding of ‘H-imipramine in human platelets. Biol. Psychiatry 20, 215-219. Briley, MS.. Raisman, R. and Langer, S.Z. (1979) Human platelets possess high-affinity binding sites for ‘H-imipramine. Eur. J. Pharmacol. 58, 347-348. Cooke, R.C. and McIntosh, J.E.A. (1986) The analysis of data from breast cancer oestrogen and progesterone receptor assays: Scatchard plots are inferior to direct fitting by computer. Clin. Chim. Acta 154, 171&180. Ellis, P.M., McIntosh, C.J., Beeston, R.. Salmond, C.E., Cooke, R.R. and Mellsop, G. (1990) Platelet tritiated imipramine binding in psychiatric patients - relationship to symptoms and severity of depression. Acta Psychiatr. Stand. 82, 275-282. Ellis, P.M., Beeston, R., Cooke, R.R. and Mellsop, G. (1991) Circadian variation in platelet imipramine binding during the day in healthy subjects. Pharmacopsychiatry (in press). Gentsch, C., Lichtsteiner. M., Gastpar, M., Gastpar, G. and Feer, H. (1989) Platelet ‘H-imipramine binding sites in depressed patients and healthy controls: a comparison between morning and afternoon samples. J. Affect. Disord. 16, 65-70. Glue, P.W.. Cowen, P.J., Nutt, D.J., Kolakowska, T. and Grahame-Smith, D.G. (1986) The effect of lithium on 5-HT mediated neuroendocrine responses and platelet SHT receptors. Psychopharmacology 90, 398-402. Goodnick, P.J., Arora, R.C., Jackman, H. and Meltzer, H.Y. (1984) Neurochemical changes during discontinuation of
110
lithium prophylaxis. II: Alteration in platelet serotonin function. Biol. Psychiatry 19, 891-898. Goodwin, F.K. and Jamison, K.R. (1990) Manic-Depressive Illness. Oxford University Press, New York, p, 31. Langer, S.Z. and Schoemaker, H. (1988) Effects of antidepressants on monoamine transporters. Prog. Neuropsychopharmacol. Biol. Psychiatry 12, 193-216. Langer, S.Z., Briley, MS., Raisman, R., Henry, J.F. and Morselli, P.L. (19801 Specific ‘H-imipramine binding in human platelets: influence of age and sex. Naunyn-Schiedeberg’s Arch. Pharmacol. 313, 189-194. Langer, S.Z., Sechter, D., Loo, H., Raisman, R. and Zarifian, E. (1986) Electroconvulsive shock therapy and maximum binding of platelet tritiated imipramine binding in depression. Arch. Gen. Psychiatry 43, 949-952. Lewis, D.A. and McChesney, C. (19851 Tritiated imipramine binding to platelets in manic subjects. J. Affect. Disord. 9, 207-211. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randal, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Maj, M., Mastronardi, P., Cerreta, A., Romano, M., Mazzarella, B. and Kemali, D. (1988) Changes in platelet ‘H-imipramine binding in depressed patients receiving electroconvulsive therapy. Biol. Psychiatry 24, 469-472. Markwell, M.A.K., Hass, S.M., Bieber, L.L. and Tolbert, N.E. (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87, 206-210. McIntosh J.E.A. and McIntosh R.P. (19801 Mathematical Modelling and Computers in Endocrinology. Springer, Berlin. Mellerup, E.T. and Plenge, P. (1988) Imipramine binding in depression and other psychiatric conditions. Acta Psychiatr. Stand. 78 (Suppl. 3451, 61-68. Mellerup, E.T., Plenge, P. and Rosenberg, R. (1982) ‘H-imipramine binding sites in platelets from psychiatric patients. Psychiatry Res. 7, 221-227. Meltzer, H.Y. and Arora, R.C. (1986) Platelet markers of suicidality. Ann. NY Acad. Sci. 487. 271-280. Mendels, J. and Frazer, A. (1975) Reduced central serotonergic activity in mania: implications for the relationship between depression and mania. Br. J. Psychiatry 126, 241-248.
Montero, D., Ofori-Adjei, D. and Wagner, A. (19891 Circadian variation of platelet ‘H-imipramine binding, platelet serotonin content and plasma cortisol in healthy volunteers. Biol. Psychiatry 26, 7944804. Muscettola, G., Di Lauro, A. and Giannini, C.P. (19861 Platelet “H-imipramine binding in bipolar patients. Psychiatry Res. 18, 343-353. Nankai, M., Yoshimoto, S., Narita, K. and Takahashi, R. (19861 Platelet [“Hlimipramine binding in depressed patients and its circadian variation in healthy controls. J. Affect. Disord. 11, 207-212. Paul, S.M., Rehavi, M., Hulihan, B., Skolnick, P.\and Goodwin, F.K. (1980) A rapid and sensitive radioreceptor assay for tertiary amine tricyclic antidepressants. Commun. Psychopharmacol. 4, 487-494. Poirier, M.F., Galzin, A.M., Pimoule, C., Schoemaker, H., Le Quan Bui, K.H., Meyer, P., Gay, C., Loo, H. and Langer, S.Z. (19881 Short-term lithium administration to healthy volunteers produces long-lasting pronounced changes in platelet serotonin uptake but not imipramine binding. Psychopharmacol. 94, 521-526. Prange, A.J., Wilson, I.C., Lynn, C.W., Alltop, L.B. and Stikeleather, R.A. (19741 r.-Tryptophan in mania. Arch. Gen. Psychiatry 30, 56-62. Rommelspacher, H. and Strauss, S. (1985) Neuroleptics and p-carbolines displace (“Hl-imipramine for its binding sites in human and rat tissue. J. Neural Transmiss. 61, 55-63. Roy, A., Everett, D., Pickar, D. and Paul, S.M. (1987) Platelet tritiated imipramine binding and serotonin uptake in depressed patients and controls. Relationship to plasma cortisol levels before and after dexamethasone administration. Arch. Gen. Psychiatry 44, 320-327. Scatchard, G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660-672. Severson, J.A., Schneider, L.S. and Frederickson, E.R. (1990) Methodological issues in the preparation and assay of platelet ‘H-imipramine binding. Psychiatry Res. 33, 19-29. Takeda, T., Harada, T. and Otsuki, S. (1989) Platelet ‘Hclonidine and ‘HI-imipramine binding and plasma cortisol levels in depression. Biol. Psychiatry 26. 52-60.
