3H-imipramine binding to freshly prepared platelet membranes in depression

JH-lmipramine Binding to Freshly Prepared Platelet Membranes in Depression Andreas E. Theodorou, Cornelius L.E. Katona, Sharon L. Davies, Anthony S. Hale, Sally M. Kerry, Roger W. Horton, John S. Kelly, and Eugene S. Paykel Received June 17, 1988; revised version received October 24, 1988; accepted January Il. 1989. Abstract. 3H-Imipramine binding was measured in freshly prepared platelet membranes from 47 drug-free major depressives and 46 healthy controls. Where possible, platelet binding in depressed subjects was repeated following treatment. A significant negative correlation was found between B~u and assay protein concentration and Bmax values were corrected for this effect. Adjusted Bmax was significantly lower (by 14%) in female depressed patients than in female control subjects, and the difference was of similar magnitude premenopausally and postmenopausally. No such difference was found in males. Kd did not differ significantly between depressed and control subjects. Multiple regression analysis confirmed significant effects on B maxof presence of depressive illness, age (positive correlation), and season (higher in summer). Within the depressed sample, BIIM was significantly lower in those subjects with obsessional features. Endogenicity (Research Diagnostic Criteria or Newcastle), dexamethasone suppression test result, drug-free interval, family history of depression, depressive psychosis, suicidal ideation, and past history of suicide attempts were not significantly related to Bmax.Paired comparisons revealed no significant effect on Bmaxof 6 weeks’ treatment with imipramine, maprotiline, or BRL 14342 or of a course of electroconvulsive therapy. Key Words. 3H-Imipramine

binding,

platelets,

depression.

Human platelets have been widely studied as an accessible model of serotonergic neurons, since they possess receptor sites and an active uptake mechanism for 5hydroxytryptamine (5HT), with closely similar pharmacological profiles to their neuronal counterparts (Stahl, 1977; Stahl and Meltzer, 1978). Kinetic studies of the active uptake of 5HT in platelets have fairly consistently reported reduced maximum velocity of uptake (a reduction in the number of uptake carrier sites) in drug-free

When this work was carried out, Andreas

E. Theodorou, Ph.D., Sharon L. Davies, B.Sc., Sally M. Kerry, M.Sc., Roger W. Horton, Ph.D.,and John S. Kelly, M.B., Ph.D., were in the Department of Pharmacology and Clinical Pharmacology; and Cornelius L.E. Katona, M.R.C.Psych., Anthony S. Hale, M.R.C.Psych., and Eugene S. Paykel, M.D., F.R.C.P., F.R.C.Psych., were in the Department of Psychiatry, St. George’s Hospital Medical School, Cranmer Terrace, London. At present, Dr. Theodorou is at the Department of Biological Sciences, North East Surrey College of Technology, Epsom, Surrey; Dr. Katona is at the Department of Psychiatry, University College and Middlesex School of Medicine, London; Dr. Hale is at the Department of Psychiatry, United Medical and Dental School, London; Prof. Kelly is at the Department of Pharmacology, University of Edinburgh; and Prof. Paykel is at the Department of Psychiatry, University of Cambridge. (Reprint requests to Dr. R.W. Horton, Dept. of Pharmacology and Clinical Pharmacology, St. George’s Hospital Medical School, London SW17 ORE, U.K.) 0165-1781/89/$03.50

@ 1989 Elsevier Scientific

Publishers

Ireland

Ltd.

88 depressed patients compared to controls (Tuomisto and Tukiainen, 1976; Coppen et al., 1978; Tuomisto et al., 1979; Meltzer et al., 1981, 1983). Studies in depressed patients receiving antidepressant medication are more difficult to interpret since direct uptake inhibition by antidepressant drugs may obscure adaptive responses. The presence of specific high affinity binding sites for 3H-imipramine (3H-IMIP) that appear, both in platelets and brain, to be associated closely with the 5HT uptake mechanism (Langer et al., 1982) provides another approach for the study of 5HT transport. Initial studies in depression reported marked reductions (29-54%) in the number of platelet JH-IMIP binding sites (Briley et al., 1980; Raisman et al., 1981, 1982; Paul et al., 1981; Suranyi-Cadotte et al., 1985~). Some subsequent studies have found less marked, though statistically significant, reductions (Lewis and McChesney, 1985; Wagner et al., 1985), and others have found no difference (Braddock et al., 1986; Carstens et al., 1986; Roy et al., 1987). Several factors may contribute to the discrepancies in these findings. Detailed methodology for platelet binding assays has varied; in particular, both freshly prepared and frozen platelet membranes, as well as intact platelets, have been used. Patients studied have varied widely in severity and symptomatology, and in the duration of the drug-free period before study. We now report the results of an investigation of platelet JH-IMIP binding in a cohort of moderately to severely depressed patients and healthy controls, in which a number of the above issues are addressed. Platelet membranes were prepared and assayed on the same day. The relationship between platelet 3H-IMIP binding and age, sex, depression, and diagnostic subgroups of depression, dexamethasone suppression test (DST), drug-free interval, seasonality, and menstrual status were investigated. In addition, it was possible in many depressed subjects to measure platelet 3H-IMIP binding repeatedly throughout a course of antidepressant treatment. Methods Selection. Depressed patients were recruited (between April 1983 and December 1985) to the study from direct referrals to an Affective Disorders Unit (both inpatients and outpatients) and inpatient transfers from acute admission wards of a psychiatric hospital serving a catchment population of 400,000. Patients were entered into the study if they fulfilled the following criteria: (I) Research Diagnostic Criteria (RDC) (Spitzer and Endicott, 1978) for major depressive disorder, probable or definite, or for bipolar depression. (2) A minimum score of 17 on the 17-item Hamilton Rating Scale for Depression (HRSD) (Hamilton, 1967). (3) No

Subject

major physical illness. (4) At least 7 days free of psychoactive drugs (apart from short-acting benzodiazepines for nighttime sedation), and at least 6 months free from depot neuroleptics. Particular efforts were made to recruit patients who were drug-free for 6 weeks or more. Controls were recruited from hospital staff and local volunteers. They were physically fit, free of psychoactive drugs, and had no past or present psychiatric illness. Psychiatric Assessments. Initial subject assessments included the following: (1) Detailed demographic, social, family, and past psychiatric history questionnaires. (An adapted version was used for controls.) (2) Ratings of depressive severity: the HRSD and the Clinical Interview for Depression (CID) (Paykel, 1985). (3) Ratings of psychopathology: the Present State Examination (PSE) (Wing et al., 1974) and the Comprehensive Psychopathological Rating Scale (CPRS) (Asberg et al., 1978). (4) Endogenicity: The Newcastle Depression Scale (NDS) (Carney et al., 1965); endogenicity was defined defined as NDS > 5. (5) Depressive diagnoses: the RDC and the International Classification of Diseases (ICD-9) (World Health Organization,

89 1977). These assessments were carried out at baseline, and HRSD and CID were repeated possible after I, 3, and 6 weeks of treatment (see below).

where

Treatment. Where possible, patients were treated for up to 6 weeks with imipramine (150 mg/ day), maprotiline (150 mg/day), BRL-14342 (a putative antidepressant of novel structure being developed by Beecham Pharmaceuticals, Harlow, U.K.; 20 mg/day), or electroconvulsive therapy (ECT, bilateral, six applications). 3H-IMIP Binding to Platelet Membranes. Thirty-ml blood samples were taken at baseline and, where possible, after 1,3, and 6 weeks of treatment by antecubital venipuncture between 9 and 10 a.m. and immediately mixed with 3 ml of acid-citrate-dextrose to prevent coagulation. Platelet-rich plasma was obtained by centrifugation at 19Og for 20 min. Platelet membranes were prepared by the method of Daiguji et al. (198 1). Aliquots of these freshly prepared platelet membranes suspended in incubation buffer (50 mMTris-HCl,pH 7.5, containing 120 mMNaC1 and 5 mM KCI) were incubated in duplicate with six concentrations (0.3-9 nM) of 3H-IMIP (specific radioactivity 20 Ci/mmole, Amersham International) for 60 min at 4 OC. Specific binding was defined as radioactivity displaced by 10-d Mdesmethylimipramine (DMI). Membrane bound radioactivity was recovered by filtration through Whatman GF/ C filters under reduced pressure. Filters were washed (4 times, 4 ml) with ice-cold incubation buffer, placed in scintillation vials containing 6 ml of Packard ES 299 cocktail, and counted at an efficiency of 3845%. Aliquots of membrane were stored at -20 ‘C before protein determination by the method of Lowry et al. (1951) using bovine serum albumen standard. The number of binding sites (BIMX)and equilibrium dissociation constants (Kd) were determined by linear regression analysis of Scatchard plots. Only those assays in which the linear regression coefficient was > 0.9 and the determined Kd was < 6 nM were considered successful and included in the results. On this basis, 23 out of a total of 194 assays were excluded. Dexamethasone Suppression Test. Dexamethasone (1 mg, p.o.) was administered to depressed patients at 8 p.m. on the same day that the baseline blood sample for platelet 3H-IMIP binding was taken. Ten ml of blood were taken by antecubital venipuncture at 3:30 p.m. on the following day, placed in a heparinized tube, and immediately centrifuged at 4000g for 4 min. Plasma was frozen at -20 “C before cortisol determination by radioimmunoassay using a commercial kit (“Amerlex,” Amersham International, U.K.). The selectivity and the reproducibility of this assay have been previously documented (Katona and Aldridge, 1985). Nonsuppression was defined as a postdexamethasone cortisol > 5 pg/dl. Statistical Analysis. BEW values approximated to a normal distribution. Kd values were skewed but approximated to a normal distribution after log transformation; statistical tests on Kd values were carried out using log-transformed data. Differences between pairs of means were compared by Student’s f test (two-tailed, unpaired except two-tailed, paired was used for the effects of treatment). For multiple comparisons, the overall differences between means were first compared using an F test on the one-way analysis of variance (ANOVA), followed by a t test (three groups) or Gabriel’s test (more than three groups).

Results Sample Characteristics. A total of 47 depressed patients and 46 controls were studied. Their demographic characteristics are summarized in Table 1. The majority of subjects in both groups were female, and most results are presented for the males and females combined and for females only; where numbers permit, results are also presented for males. Clinical features of the depressed subjects are shown in Table 2. All but 6 were inpatients, 43 had unipolar depression, and 4 had bipolar depression. All had moderate to severe depression (HRSD score range 17-45).

90 Table 1. Demographic

details of control and depressed

subjects

Controls

Depressed

46

47

Total number Females Premenopausal

32

34

23

21

9

13

Postmenopausal Males

14

13

Age (all)

41 .o IL 12.2

45.0 k 14.4

Females

39.9 + 11.3

43.2 f 14.0

Premenopausal

35.4 F

a.9

33.1 +

5.8

Postmenopausal

52.8 +

6.6

59.1 k

7.6

43.2 f

9.0

49.8 f

a.7

Males Note.

Ages are means t SD.

Table 2. Clinical

RDC definite Newcastle

endogenous

endogenous

DST nonsuppressorsl Baseline

features of depressed

HRSD total

% %

%

subjects

Total

Female

Male

(n = 47)

(n = 34)

(n = 13)

72

74

69

55

47

77

48

43

67

24.8 f 5.5

24.1 + 5.2

26.7 f 7.2

Note. RDC = Research Diagnostic Criteria. DST = dexamethasone suppression test. HRSD = Hamilton Rating Scale for Depression. HRSD total is the mean i SD scores on the 17-item HRSD (Hamilton. 19671. 1. The DST was carried out in 21 females and 6 males.

Our initial analysis showed a statistically significant correlation between Bmaxof 3H-IMIP binding and platelet membrane protein concentration. This held true for controls (I = -0.44, n = 46,~ < 0.0 I), depressed subjects at baseline (I = -0.56, n = 47,~ < O.OOl), and for all assays from depressed subjects during treatment (r = -0.42, n = 78, p< 0.001). Simple linear regression provided an adequate model for this relationship; the addition of a quadratic term did not result in a significant improvement in fit. Slopes of the linear regression lines did not differ significantly between males, premenopausal females, or postmenopausal females or between baseline assays and those carried out during treatment. The relationship between Bmax and protein is given by the equation Bmax = 2364 - (10.31 x protein, pg/sample). There was no overall difference in platelet membrane protein between control and depressed subjects in the total, female, or male samples (Table 3). Variations in platelet protein concentration between individuals, however, may have had considerable effects on Bmaxestimations. Therefore, individual Bmaxvalues were corrected for the effect of platelet protein using the derived equation: Adjusted Bmax (AdjBmax) = Bmax + (10.31 x protein) + K. The numerical value of K does not affect comparisons between groups; Adj Bmaxis reported using K = -764 (the difference between 2364 and the mean measured Bmax, 1600), so that the AdjBmx values fall within the range of measured BmaL Results are presented for both Bmax and AdjBmax in Tables 3-6 and Table 9.

91 Table 3.3H-lmipramine

binding parameters in control and depressed subjects Protein

Kd

Bmax

AdiBmax

Controls (n = 46)

75.0 f 25.8

0.96 k 0.47

1668 k 441

1680 + 400

Depressed (n = 47)

72.9 f 26.7

1.11 * 0.75

1538 k 548

1527 + 439

Controls (n = 32)

78.2 AI 25.5

0.92 f 0.28

1713 k 475

1758 + 413

Depressed (n = 34)

75.8 f 29.7

1.17 f 0.87

1492 f 571

1510 Liz4371

Controls (n = 14)

67.9?

16.8

1.04 f 0.79

1564 f 348

1503 f 2992

Depressed (n = 13)

65.3 + 16.2

0.97 + 0.22

1658 f 480

1571 f 480

All subjects

Females

Males

Note. Protein @g/assay); Kd Wf); Bmax and AdjSmax number of subjects shown in parentheses. 1. Significantly 2. Significantly

different different

from female controls: from female controls:

(fmol/mg

p < 0.05. p -C0.05 (Gabriel’s

protein).

Values are means k SD for the

test)

Comparisons Between Depressed Subjects (Pretreatment) and Controls. Bmx, AdjBmax and Kd did not differ significantly between the total depressed and control groups (Table 3). Adj Bmax was significantly lower (14%) in depressed female subjects compared to controls. A similar (13%) reduction in unadjusted Bmx was apparent in depressed females but just failed to reach statistical significance. The reduction in AdjBmax was of similar magnitude premenopausally (12%) and postmenopausally (15%). Within premenopausal subjects, the reduction was apparent when blood sampling was performed in either the preovulatory (9%) or the postovulatory (13%) phase of the menstrual cycle. The Kd of JH-IMIP binding did not differ significantly between female depressed patients and controls. Bma$ Adj Bmaxand Kd did not differ significantly between the male depressed patients and controls. Comparisons Between Depressive Subgroups and Controls. There were no significant differences either within the total sample or the female subjects in Adj Bma3 Bma+ or Kd between endogenous and nonendogenous subgroups defined by either RDC or Newcastle criteria (Table 4). Newcastle endogenous subjects were significantly older than Newcastle nonendogenous subjects and controls, both in the total and in the female groups. Using Newcastle or RDC definitions, endogenous and nonendogenous subgroups did not differ significantly from controls in Adj Bmax, Bm+ or Kd, either in the total group or the female subjects (Table 4).

92 Table 4. 3H-lmipramine binding defined depressed patients

parameters

in controls

and diagnostically

Me

Kd

Bmax

AdjBmax

Controls rn = 461

41.0 t 12.2

0.96 + 0.47

1668 ?I 441

1680 ? 400

RDC end In = 34~

44.5 k 14.6

1.16 i 0.82

1552 + 612

i 530 2 484

RDC non-end in = 13~

46.3 k 14.1

0.98 + 0.32

1495 f 328

1508 2 346

Newcastle end rn = 261

49.0 ?I 14.31

1.172

0.92

1586 k 648

1528 + 670

Newcastle non-end In = 21~

40.1 k 12.4

1.03 ? 0.37

1510 t 422

1490 k 399

Controls In = 321

39.9 t 11.3

0.92 4 0.28

1713 i 475

1755 -t 413

RDC end cn = 25~

42.9 k 14.0

1.21 + 1.00

1505 t 645

1521 i 470

RDC non-end in = 91

44.0 ?I 15.0

1.03 2 0.39

1448 t 312

1469 i 354

Newcastle end in = 16,

48.7 k 14.81

1.28 k 1.20

1476 rt 724

1529 i- 532

Newcastle non-end in = 18)

38.3 k 11.9

1.06 I? 0.38

1502 + 412

1487 k 340

All subjects

Female subjects

Note. Kd I~MI; Bmaxand AdjEmax ifmol/mg protein). Valuesare meanstSD. Binding parametersdid not differ significantly between depressed subgrouppairsor between subgroupsand controls. RDC = Research Diagnostic Criteria. End = endogenous. Non-end = nonendogenous. 1. Newcastle endogenous jects: p < 0.05.

subjects

were significantly

older than Newcastle

nonendogenous

and control

sub-

Dexamethasone Suppression Test (DST). DSTs were carried out in 27 depressed subjects. DST nonsuppressors were older than DST suppressors, but did not differ significantly in Bma$ AdjBmax, or Kd of 3H-IMIP binding, in the total or the female group (Table 5). DST nonsuppressors had significantly lower Bmx values than conin the total sample and 23% for Bmaxand 22% trols (2 1% for Bmaxand 19% for Adj B max for AdjB,,, in the female sample). A similar, though not statistically significant, reduction (13% for Bmx and 10% for Adj B maxin the total group and 20% for Bmaxand 15% for Adj Bmaxin the females) was apparent comparing DST suppressors and controls. There were no statistically significant correlations between postdexamethasane cortisol concentration and Bmax,AdjBmax, or Kd within the total group of depressed patients or the females. Specific

Clinical

Features.

Depressed

subjects

were divided

by past history

of

93

suicide attempts and serious suicide attempts and current suicidal intent (HRSD), obsessional features (HRSD and CID), diurnal mood variation (HRSD and CID), weight loss (HRSD), reactivity of mood (CID), psychosis (CID delusions and RDC definite psychotic depression), and family history of depression or other psychiatric illness. Correlations between severity of depression (total HRSD scores) and binding parameters were also examined. Table 5.3H-lmipramine

binding and the dexamethasone suppression test Kd

Me

B Inax

AdpMX

All subJe&s Controls in = 46)

41.5

4

12.2

0.96 rt 0.47

1668 I: 441

16813 t: 400

suppressors (ff = 33)

36.5 I

9.7

0.9-I + 0.25

1453 1.371

1513 t 267

47.6 -i- 12.3

1.00 + Lx34

1333 z!z404~

1506 rt 3112

39.9 4 17.3

0.92 + 028

-In3

rt 475

1755 It 413

34.6 I

7.5

0.66 i 0.33

t 370 rt 420

3463 k 3m

46.7 4 14.2

0.99 r 0.37

1322 + 4fS2

$371 I!z 3671

DST

nonsuppres-

5ST sors

in =

14)

f%m8leoub@&s Controfs [n = 32) DST

suppressors

@? =

DST

91

nonsuppres-

sors

fn =

12)

No&. Kd InM); 3max and Adjflmax means I SD.

(fmolimg protein!. DSf

= daxamWthaSw%? SkippfwSion

test. Values

are

1. Significantly different from controts. p -= D.Of. 2. Signiflcantfy different from controls, p c 0.05.

Subjects with obsessional features (HRSD ~bsessionality item 2 t) had significantly fower Bmaxvalues (1306 * 408 fmol\ mg protein, n = 221, but unaltered red, compared to depressed patients without obsessional symptoms (i 739 f 580 fmoI/ mg protein, n = 25, p < O.Qt). The same pattern was apparent for Adj&ax (obsessional 1379 + 338, nonobsessional 1651 f 495,~ < 0.05). When the more stringent criterion of CID obsessionaiity score Z 4 (indicating obsessional ideas and rumination of at least moderate intensity) was used, this finding held true (Adj&sx obsessionals 1283 * 326 fmol/ mg protein, n = 12, nonobsessionals 1596 * 454 fmoli mg protein, tt = 34, p < 0.05). Depressed patients with obsessional features also showed significantly lower B,sX and AdjBman than controls. A positive correlation was found between HRSD scores and AdjBmax(r = 0.33, p < 0.05), but not BRIM (r = 0.23, NS). Restricting the analysis to females only revealed a similar pattern. With either HRSD or CID definitions of obsessionality, subjects with obsessional features had significantly lower Bmax(CID definition, Ii86 f 285, n = 10 vs. 1601 f 623 fmol/mg protein, n = 23, p < O.OS), but the reduction in Adj Bmax (1334 f 29 1 vs. 1563 f 475 fmol/ mg protein) did not reach statistical significance. Female patients with a past history of serious suicide attempts showed a lower Kd (0.82 4 0.05 nM, n = 12~s. 1.35 f

94 0.22 nM, n = 22, p < 0.05) than those without such a past history. There was no significant correlation between HRSD score and AdjBmax or Bmax. B mx, Adj Bmax, and Kd did not differ significantly between groups defined by any of the other clinical variables (current suicidal intent, diurnal mood variation, weight loss, reactivity of mood, psychosis, or family history) in either the total group or the female subjects. Drug-Free Interval. Depressed subjects were divided according to the length of time (7-2 1 days [n = 161 and 22 days or more [n = 3 11) since last exposure to antidepressants or to major tranquilizers (Table 6). There were no significant differences between these two groups in Bmaxor Adj Bma%although Kd values were significantly lower in subjects with the shorter antidepressant-free interval. Bmax and Adj Bmax of depressed patients antidepressant-free for 7-21 days and for 22 days or more did not differ significantly from controls. The length of the neuroleptic-free interval was not significantly related to 3H-IMIP binding parameters. Table 6. Effects of drug-free depressed subjects

interval

on the binding

of 3H-imipramine

Bmax

AdjBmax

0.87 + 0.201

1339 k 476

1515 f 340

45.7 + 14.5

1.24 + 0.89

1638 + 562

1528 + 496

7-21 days (n = 6)

44.2 -t 14.2

0.84 2 0.20

1352 f 473

1530 IL 407

22+ days (n = 41)

45.1 + 14.1

1.15 + 0.77

1563 + 557

1523 F 455

Age

Kd

7-21 days in = 161

43.7 IL 14.0

22+ days (n = 31)

in

Antidepressant-free

Neuroleptlc-free

Note. Kd (nhf); Bmax and AdjSmax 1. Significantly

different

(fmol/mg

protein ). Values are means i- SD.

from 22+ days, p ,: 0.05

Age and Sex. Age did not correlate significantly with Bmax or Adj Bmax of 3H-IMIP binding in control or depressed patients. Bmax, though not AdjBmax, did correlate significantly with age (r = 0.20, p < 0.05), when results from control and depressed subjects were combined. There was also a significant correlation between age and Kd in all depressed subjects (r 0.25,p< 0.05) and female depressed subjects (r = 0.29,~ < 0.05). No significant correlation was evident for controls. Control males had significantly lower AdjB,,, of 3H-IMIP binding than control females (Table 3). This did not reach statistical significance for Bmax. There was no significant difference in binding parameters between male and female depressed subjects. q

Season. Subjects were divided by season of testing. Adj Bmax values for control and depressed subjects are shown in Table 7. In control and depressed subjects combined, Adj Bmaxwas significantly higher in summer than winter (Table 7). In samples taken in the summer, females had significantly higher AdjBmax than males (Table 8). Multivariate

Analysis.

To examine

the effects of possible interrelationships

between

95 Table 7. Seasonal variation and depressed subjects

in AdjB mr~of platelet 3H-imipramine

in control

All

Controls

1529 f 393 (n = 21)

1735 + 389 (n = 8)

1401 f 350 (n = 13)

1759 f 5831 (n = 24)

1895 2 585 (n = 7)

1704 + 598 (n = 17)

Autumn (Sept-Nov)

1823 2 445 (n = 19)

1736 2 484 (n = 11)

1466 f 344 (n = 8)

Winter (Dee-Feb)

1504 2 253 in = 291

1545 f 235 (n = 20)

1411 + 285 in = 91

Season Spring (Mar-May)

Summer (June-Aug

)

Dewessed

Note. Values are means ? SD of AdjSmax (fmol/mg protein 1. Analysis of variance revealed significant effects of season (F = 3.46; df = 3, 80; p -Z 0.05) and illness (depressed vs. control, f = 7.57; df = 1. 80; p < 0.01). and significant interactions between season and sex (F=4.42; df=3.8O;p~O.O1 )and illnessandsex (F=8.54;df=l, 80; p < 0.01). 1. Significantly different from winter, p c 0.05.

protein, age, sex, season, menopausal status, and illness on &ax of 3H-IMIP binding, a multiple regression analysis was performed. The results revealed significant effects of protein(F=29.14;d~=1,86;p<0.001),illness(F=8.17;df=1,86;p<0.01),age(F= 5.32; df = 1, 86; p < 0.05), and season (F= 2.71; df = 3, 86; p < 0.05). The effects of protein, age, sex, season, and menopausal status explained 34.1% of the total variance, and this was increased to 39.9% by including illness. The following clinical variables were then each added to the multiple regression model: obsessionality, suicidal intent and past history, drug-free interval, severity (HRSD), and endogenicity (Newcastle and RDC). Only obsessionality (using HRSD item, F= 6.92; df = 1,84;p
Males

in male and

Females

Spring (Mar-May)

1611 + 358 (n = 9)

1486 k 421 (n = 12)

Summer (June-Aug)

1410 + 574 (n = 8)

1934 f 5141 (n = 16)

Autumn (Sept-Nov)

1647 f 392 (n = 2)

1620 f 463 (n = 17)

Winter ( Dee-Feb)

1538 + 196 (n = 8)

1491 f 273 (n = 21)

Note. Values are means ? SD of AdjBmax (fmol/mg protein). 1. Significantly different between males and females, p < 0.01.

96 Effects of Treatment and Improvement. There was no significant difference in baseline Bmax or Kd of 3H-IMIP binding between those depressed patients who responded favorably to treatment within 6 weeks (final HRSD score < 10, or a reduction from baseline HRSD score > 50%) and those who did not. However, baseline AdjBmax tended to be lower in treatment responders (1262 +- 373 fmol/ mg protein, n = 8) than nonresponders (1515 f 482 fmol/mg protein, n = 17). The effects of treatment were examined using paired comparisons for individual patients between samples taken at 0, 1,3, and 6 weeks of treatment with imipramine (n = S), maprotiline (n = 9), BRL-14342 (n = 5), or after six applications of ECT (n = 7). No treatment-related differences in B maxor Adj Bmaxwere found (Table 9) either in the total patient group, or when subjects receiving ECT were excluded and when subjects were subdivided into treatment responders and nonresponders. Kd was significantly increased in the total patient sample after 1 week of treatment, but not at other times (Table 9). A similar nonsignificant trend was apparent after 1 week when patients receiving ECT were excluded. Table 9. Effects of treatment with baseline values Week

0

Week

1

Week

0

Week

3

Week

0

Week

6

Excluding Week

0

Week

1

Week

0

Week

3

Week

0

Week

6

on 3H-imipramine

Bm.x

Kd

23

0.95 !z 0.48

1516+

1.11 rh 0.481

1405+364

1461 k 317

1.01 + 0.48

1410 * 619

1430 2489

1.21 * 0.77

1290?326

1465 5379

1.00 + 0.52

14582680

1548 +464

1.04 +_ 0.36

1349+324

1471 2284

0.89 i 0.32

1451 1384

1505 k 268

1.02 + 0.28

1431 f404

1520 t336

0.96 f 0.33

1305+396

1364f326

1.10 + 0.62

1250f313

1475 +392

1301 + 402

1499 i- 228

1371 z! 360

1546k

16

patients treated with electroconvulsive 16

17

12

Note. Kd (nM); Bmax and Adj8max comparisons indicated in).

0.90 t 0.28

ifmol/mg

proteini.

590

1526+451

therapy

1.07 +_ 0.38

different

AdjBmax

n

23

1. Significantly

Bmax- paired comparisons

Values

are means

& SD for the number

256 of paired

from paired values at week 0, p < 0.05.

Discussion Differences Between Depressed Patients and Controls. Our results indicate that the number of 3H-IMIP binding sites is slightly (9%) but not significantly, lower in the total group of depressed patients compared with age-matched controls. The magnitude of this difference is similar to that reported in several recent studies (Wagner et al., 1985; Baron et al., 1986; Carstens et al., 1986; Muscettola et al., 1986; Roy et al., 1987). In only one of these studies was the difference statistically significant (Wagner et al., 1985).

97 We did, however, find a significant reduction (14%) in the number of JH-IMIP binding sites in female depressed patients. Most previous studies have not found sex-related differences, although a similar reduction in JH-IMIP Bmx in depressed females, but not males, was recently reported by Roy et al. (1987). We also found that in control, but not depressed subjects, females had significantly higher Bmax values than males. Roy et al. (1987) reported a similar, though not significant trend, whereas other studies have found no differences between females and males (Wagner et al., 1985; Carstens et al., 1986). Hormonal factors may contribute to the differences we have observed between males and females. However, we found that the magnitude of the reduction in female depressed patients was similar when examined in premenopausal or postmenopausal subjects, and in the former group in the preovulatory and postovulatory phases of the menstrual cycle. This is consistent with the finding that Bmaxof 3H-IMIP binding in controls is not significantly altered throughout the. menstrual cycle (Poirier et al., 1986~). The magnitude of the difference in JH-IMIP Bmaxbetween depressed and control subjects is much smaller than that reported in several earlier studies (Briley et al., 1980; Paul et al., 1981; Raisman et al., 1981; Suranyi-Cadotte et al., 1985~) and some recent studies (Poirier et al., 19866; Langer et al., 1986). Subject selection may be important. Several earlier studies relied on clinical diagnoses, and though more recent articles, like ours, have used operational diagnostic criteria, most have nonetheless examined heterogeneous samples of depressed patients. Methodological Aspects. Although widely used, DMI may be less than ideal to define 3H-IMIP binding to the 5HT transporter. Studies in rat brain indicate that DMI displaces 3H-IMIP from additional sites not associated with 5HT transport (D’Amato et al., 1987). However, our use of DMI does allow direct comparisons to be made with previous studies. Differences in absolute values of B maxof 3H-IMIP binding have varied as widely as differences between depressed and control subjects. Reported mean control values have ranged from 450 to 2656 fmol/mg protein (Paul et al., 1981; Schneider et al., 1985). This variation may reflect differences in platelet preparation, storage, and assay procedure. Our values are at the higher end of the range. Unlike most studies, we have prepared and assayed platelet membranes within hours of blood sampling and with no intermediate freezing or storage. It is noteworthy that other studies (e.g., Tang and Morris, 1985; Innis et al., 1987) using freshly prepared platelet membranes also report relatively high control (>lOOO fmol/mg protein) 3H-IMIP Bmax values. This might suggest that JH-IMIP binding sites are lost on freezing or storage. However, Lewis and McChesney (1985) reported no significant differences in 3H-IMIP Bmax attributable to freezing. Anderson et al. (1984) found low Bmax of 3H-IMIP binding in unwashed previously frozen platelet membranes (690 fmol/mg protein), which was increased almost two-fold by washing, probably as a result of removal of endogenous SHT. Our high values may thus be attributable to our procedure of using well-washed freshly prepared platelet membranes. We are currently completing a study of a new sample using frozen platelet membranes which will enable this question to be examined further. Platelet protein concentration in the assay may also be relevant. Mellerup et al.

98 (1982) have highlighted the wide variation in membrane protein concentration used in the analytical procedures, ranging from 170 to 2500 lg/ml , and argued that low protein concentrations give rise to more accurate determinations of Bmaxand Kd. We have used a protein concentration of approximately 75 I.cg per sample (150 pg/ ml), similar to that used by Mellerup et al. (1982). We found a significant negative correlation between membrane protein concentration and 3H-IMIP Bmaxacross control and depressed subjects. This is similar to, but of greater magnitude than, the finding reported by Arora et al. (1985), although lnnis et al. (1987) found no such significant correlation. We therefore adjusted Bmaxvalues to eliminate the effects of variable protein concentration, using a strategy similar to that adopted by Arora et al. (1985). The possible confounding effect of assay protein concentration has not been taken into account in most previous studies of 3H-IMIP binding in depression. The relationship between protein concentration and Bmax we have demonstrated, and the correction we have used may not be applicable to other studies, particularly since we have used fresh membranes and most others have studied frozen membranes. However, we suggest that future studies should take this factor into account. Other Correlates. We found a summer peak in the Bmaxof 3H-lMlP binding in both depressed and control subjects. There was a significant interaction between sex and season, with the seasonal variation restricted to female subjects. No consistent findings have emerged from previous studies of seasonal effects. Retrospective studies in control and depressed subjects have shown no seasonal difference (Carstens et al., 1986) a winter trough (Hrdina et al., 1985), and a winter peak (Kanof et al., 1987). Prospective studies of serial samples from controls have shown no seasonal differences (Tang and Morris, 1985; Galzin et al., 1986) a summer peak (Egrise et al., 1986; Arora and Meltzer, 1988), and a summer trough (Whitaker et al., 1984). Our findings are consistent with the study of Egrise et al. (1986), who also examined depressed patients prospectively and found a summer peak, as in controls. The interaction we report between sex and season has not been examined in other studies and may thus contribute to the lack of consensus on seasonal variation. Our finding of a modest, but significant positive correlation between age and Bmaxis in keeping with the higher values in elderly than younger control subjects (Schneider et al., 1985). Previous antidepressant treatment may influence baseline measures of 3H-IMIP binding in depressed patients. The drug-free interval has varied widely between studies. Several studies have attempted to quantify the effects of the drug-free interval in healthy controls. 3H-IMIP Bmaxwas found not to differ from baseline values 2 weeks after discontinuation of 3 weeks’ administration of imipramine (Suranyi-Cadotte et al., 19856) or 1 week’s exposure to maprotiline (Poirier et al., 1987); tended to be reduced 2 weeks after 1 week’s administration of amineptine (Poirier et al., 1987); but was reduced for up to 4 weeks following 1 week’s exposure to clomipramine (Poirier et al., 1987) and increased for up to 4 weeks following 18 days’ administration of amitriptyline (Braddock et al., 1984). The effects of previous drug treatment appear to depend on the specific drug involved and may be related both to persisting drug effects and to drug withdrawal. Depressed patients in our study varied widely in the drug-free

99 interval and also in recent drug history. Although B maxvalues tended to be lower in those subjects drug free for < 3 weeks compared to those drug free for longer intervals, this tendency was no longer apparent after correction for assay protein concentration. Assay protein concentration may thus also contribute to the previously reported effects of the drug-free interval. Heterogeneity of clinical features within depressed patients may have an important influence on overall comparisons between depressed patients and controls, and could explain differences between studies. Several studies have compared clinically defined subgroups of patients. In agreement with the present findings, no differences in 3H-IMIP Bmaxhave been reported between endogenous and nonendogenous unipolar depressed patients (Baron et al., 1986; Roy et al., 1987). Both retarded depressed patients (Carstens et al., 1986) and, in contrast to our findings, psychotic (Arora et al., 1985) depressed patients had lower 3H-IMIP binding Bmaxthan other depressed patients or controls. A positive family history of depression was associated with reduced JH-IMIP Bmaxin some studies (Lewis and McChesney, 1985; Schneider et al., 1986), but our findings support the absence of such a relationship as reported by Carstens et al. (1986). Although a negative correlation between severity of depression and 3H-IMIP binding Bmaxhas been reported in two studies (Tanimoto et al., 1985; Nankai et al., 1986), most studies (e.g., Raisman et al., 1981; Baron et al., 1986) found no such relationship. We found a weak but significant positive correlation between HRSD scores and 3H-IMIP binding AdjB maxin the total sample. This should be interpreted with caution since no such relationship was apparent when the analysis was restricted to female depressed patients. Previous suicide attempt has not been associated with altered 3H-IMIP Bmnx(Roy et al., 1987), although Wagner et al. (1985) reported a tendency toward a higher B WC in violent compared to nonviolent suicide attempters. Although we found no significant relationship between 3H-IMIP binding Bmaxand past history of suicide attempt, female subjects who had made serious attempts on their lives showed higher binding affinity than those without such a past history. We found depressed patients with obsessional features to have significantly lower B,IWand Adj Bmaxvalues than either controls or depressed patients without obsessional features. This is consistent with the finding of reduced JH-IMIP binding Bmaxin primary obsessional illness (Weizman et al., 1986) and the favorable response of patients with obsessional illness to selective 5HT uptake inhibitors reported in several studies (Montgomery, 1980; Thor& et al., 1980; Kahn et al., 1984; Turneret al., 1985; Flament et al., 1987; Price et al., 1987). To our knowledge, no other study has examined 3H-IMIP binding in relation to obsessionality within depression. Further investigation is clearly required to determine whether obsessional symptoms identify those depressed patients with reduced JH-IMIP binding Bmaxor whether our results are due to chance. We found no overall relationship between DST response and 3H-IMIP binding B~FCG, although female nonsuppressors had significantly lower Bmaxvalues than controls. This should be interpreted with caution since it was not possible for the DST to be performed in all our depressed patients, and both DST suppressors and nonsuppressors had relatively lower B max values than the depressed group as a whole. Our findings are consistent with those of Roy et al. (1987), who found DST non-

suppression to be associated with reduced B maxin females only, and Carstens et al. (1986), who found no overall differences between controls and depressed patients in respect of DST result. Research to date has not clarified whether baseline JH-IMIP binding in depression predicts subsequent treatment response. A tendency for treatment responders to have higher initial 3H-IMIP BW values was reported by Wagner et al. (1987) and a similar, statistically significant result was found by Hrdina et al. (1985). In contrast, Schneider et al. (1986) reported significantly lower initial 3H-IMIP Bmaxin treatment responders than in nonresponders. These discrepant results may be a reflection of variability in adequacy of treatment (particularly duration) and response rate. Our results are in keeping with those of Schneider et al. (1986) although the proportion of treatment responders (32%) in our study was relatively low. This may also reflect the treatmentresistant nature of patients referred to a specialist unit for affective disorders. Several studies (Asarch et al., 1981; Raisman et al., 1981; Braddock et al., 1986; Schneider et al., 1986) as well as our own, have found that short-term (up to 8 weeks) antidepressant treatment did not significantly alter Bmaxof 3H-IMIP binding. Most of these studies, like ours, used a variety of treatment modalities. This may be of particular importance. Langer et al. (1986) found that a short course of ECT resulted in a significant increase in Bma% though values remained below those of controls. Wagner et al. (1987) reported an increase in B rna~after 3 weeks of treatment but found significant differences between treatment groups-the increase being virtually restricted to patients receiving zimelidine and alaproclate. Results of long-term treatment and of recovered patients have been more equivocal. Wagner et al. (1987) found no change from pretreatment values at l- and 2-year followup, but normalization of initially reduced B maxafter cessation of successful treatment was reported by Suranyi-Cadotte et al. (1982). Thus, though Bmax of 3H-IMIP binding does not normalize simultaneously with clinical improvement, it may do so after some delay. It is not therefore possible at present to regard 3H-IMIP binding Bmaxas unequivocally either a state or a trait marker for depression. Acknowledgment. This study was supported

by a Major Award from the Wellcome

Trust.

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