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Platelet serotonin markers and depressive symptomatology
i
Platelet Serotonin Markers and Depressive Symptomatology Yvette I. Sheline, Mark E. Bardgett, Jamie L. Jackson, John W. Newcomer, and John G. Csernansky
Dysfunction of brain serotonergic symptoms may be a factor in the mood and behavioral disturbances associated with depression. Platelet serotonin measures represent indirect but easily obtainable indices of brain serotonin function. To examine the specificity, of relationships between cognitive and vegetative symptom groupings and platelet serotonin measures, we assessed 35 depressed outpatients using the Hamilton Rating Scale for Depression and collected platelets after a minimum 3-week drug-free period. Platelets were also collected from 14 controls. The results showed that depressed patients had lower platelet serotonin (5-HT) uptake site density values than controls and that 5-HT uptake site density values were inversely correlated with the severity, of cognitive symptoms of depression. Platelet 5-HT2 receptor density values were higher in depressed patients than controls, and there was a trend toward a direct correlation between the cognitive symptoms of depression and 5-HTz receptor density' values. Neither platelet measure showed any relationship with the severity of the vegetative symptoms of depression. Key Words: Depression, serotonin, platelets, 5-HT uptake,
Introduction There is growing evidence that dysfunction of serotonergic systems may be an underlying factor in the pathophysiology of mood disorders (Meltzer and Lowy 1987). Among the more direct measures of central nervous system (CNS) serotonergic function is cerebrospinal fluid (CSF) concentration of 5-hydroxyindoleacetic acid (5-HIAA) concentrations, the principal metabolite of serotonin (5-HT). Depressed patients with melancholia have lower concentrations of CSF 5-HIAA than normal controls, and depressed From the Department of Psychiatry, Washington University School of Medicine, St. Louis, M163110. Address reprint requests to Yvette Sheline. MD, Assistant Professor of Psychiatry,, Department of Psychiatry, Washington University School of Medicine, 4940 Children's Place, St. Louis, MO 63110-1093. Received March 11, 1994; revised June 1, 1994.
© 1995 Society, of Biological Psychiatry
5 - H T 2 receptor
patients who make suicide attempts have lower CSF 5HIAA concentrations than other depressed patients (Asberg et al 1984). Further, increases in CSF 5-HIAA have been found after recovery from depression (Traskman et al 1981). In one follow-up study, patients with lower CSF 5-HIAA concentrations during an episode of depression were more likely to re-attempt suicide within 5 years (Roy et al 1989). Platelet 5-HT uptake sites and 5-HT2 receptors may provide indirect, but readily accessible, measures of serotonin activity. Previous studies have demonstrated decreases in platelet 5-HT uptake site densities and increases in platelet 5-HT2 receptor densities in unmedicated, depressed patients compared to normals (Paul et al 1981; Biegon et al 1987). Some investigators have reported the density of uptake sites to remain decreased after recovery in unipolar depressed 0006-3223195/$09.50 SSDI 0006-3223(94)00160-5
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patients (Coppen et al 1978; Scott et al 1979; Healy et al 1990). Others, however, have found that both 5-HT uptake site density decreases and 5-HTz receptor density increases associated with depression tend to normalize after treatment with antidepressants (Wagner et al 1987; Freeman et al 1993; Biegon et al 1990b). Although there may be differences in platelet 5-HT uptake site and 5 HT2 receptor density values between depressed patients and controls, it remains unknown to what extent these alterations underlie specific dimensions of mood and behavioral pathology in depression (Apter et al 1990). For this reason, we decided to examine correlations between platelet serotonin markers and the two major groupings of depressive symptoms, "cognitive" and "vegetative" symptoms. Prior factor analytic studies (Rhodes and Overall 1983) have identified cognitive and vegetative superfactors composed of items in the Hamilton Rating Scale for Depression (HRSD) (Hamilton 1960). Cognitive symptoms denote the items in the (HRSD) that affect mental functioning, such as mood and guilt, whereas vegetative symptoms denote items reflecting somatic functioning. Given the association between 5-HT abnormalities and anxiety, aggression, impulsivity, mood instability, and suicide in a variety of psychiatric disorders in addition to depression (Apter et al 1990; Csernansky and Sheline 1993) we hypothesized that platelet 5-HT markers would be correlated with cognitive symptoms of depression but not with vegetative symptoms. In an attempt to replicate prior work we also hypothesized that depressed patients would show lower 5-HT uptake site density values and higher 5-HT2 receptor density values compared to the control group.
Methods
PatientSample Patients were recruited through local newspaper advertisements to participate in controlled antidepressant drug trials, as well as this research protocol. All patients gave written informed consent prior to their participation. There were 13 men and 22 women who participated. Mean age was 44.1 _+ 14.5 (SD), range = 18-77. All patients were interviewed by board-certified and specially trained research psychiatrists (YIS and JGC) using a semi-structured clinical interview. All patients met DSM-III-R criteria for unipolar major depression at baseline. Exclusionary criteria included other primary DSM-III-R diagnoses, recent drug or alcohol abuse or dependence, major medical illnesses, and the use within the past 3 weeks of any drugs affecting central nervous system (CNS) function such as psychotropic drugs, calcium channel blockers, and [3 blockers. Symptoms were assessed using the 17 item HRSD (Hamilton 1960), which was administered after a 7-10 day placebo washout. Mean HRSD score was 22.9 -+
6.5 (SD). The range of values was 2-41, indicating that the symptoms of some patients were changed considerably during the placebo wash-out. Two groupings of depressive symptoms were assessed using the vegetative and cognitive HRSD superfactors identified by Rhoades and Overall (1983). The vegetative superfactor included sleep disturbance (early, middle, and late), somatic anxiety, gastrointestinal (GI) symptoms, general somatic symptoms, genital symptoms, hypochondriasis and weight loss (Items 4-6, 11-16). The mean vegetative superfactor score was 10.7 -+ 3.5 (SD), range 1-18. The cognitive superfactor included depressed mood, guilt, suicidal ideation, work/activities, retardation, agitation, psychic anxiety, and insight (Items 1-3, 7-10, 17). The mean cognitive superfactor score was 12.2 + 4.0 (SD) range 0-18.
Control Sample A sample of 14 subjects matched for age, gender, and socioeconomic status was recruited through advertisements. Mean age for the sample was 40.4 _+ 9.8 (SD), range 24-59, and included 10 women and 4 men. Control subjects were included if they had no current or previous DSM-III-R psychiatric diagnosis. In addition, control subjects were subject to the same drug exclusionary criteria as experimental subjects.
Platelet Samples Platelets were prepared according to a standard protocol as follows: 15 ml of blood were collected into vacutainers containing 0.3 ml of 7.5% ethylenediamine tetracetic acid (EDTA) (pH 7.5). Platelet-rich plasma (PRP) was obtained by centrifugation of the blood at 900 rpm for 10 min at 22 ° C in an IEC Centra-8R centrifuge. The PRP was removed using a plastic pipette, divided into three tubes, and recentrifuged at 3400 rpm for 15 min. Then the supernatant was separated from each platelet pellet. The pellet was resuspended in 2 ml of phosphate-buffered saline glucose (pH 7.2) and centrifuged again at 3400 rpm for 15 rain. After discarding the supernatant, the platelet pellets were immediately frozen at - 7 0 ° C for storage until analysis.
5-HT2 Receptors (~25I-LSDAssay) Randomly selected platelet pellets from each subject were slowly thawed and resuspended in 5 ml of 5 mmol/L Tris containing 0.1% EDTA (pH 7.5) and homogenized using a teflon pestle and glass homogenization tube. The homogenate was then centrifuged at 50,000 g for 15 min at 4 ° C. Each pellet was again re-suspended in 2.5 ml ice-cold incubation buffer (50 mmol/L Tris, 120 mmol/L NaCI, 5 mmol/L KC1, 2mmol/L MgC1, 0.05% ascorbic acid, pH 7.3) and rehomogenized.
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Protein concentrations were determined using the BioRad Protein Assay using bovine gamma globulin as the standard. Specific binding of lzSI-LSD (NEN Dupont) was determined at six-ligand concentrations (0.06-1.2 riM) in the presence and absence of 1 Ixmol/L spiperone (Research Biochemicals, Inc.). This method was developed in a series of initial exploratory studies to determine the range and number of ligand concentrations necessary to obtain highly reliable linear Scatchard transformations with a minimum of sample. Incubations were performed in microcentrifuge tubes and contained 80 txl of platelet suspension (10 p~g protein), 10 Ixl incubation buffer or spiperone and 10 ixl 12SI-LSD. Tubes were incubated at 37 ° C for 2 hr. The incubations were stopped by the addition of 1 ml ice-cold 4 ° C Tris/BSA buffer (50 mmol/L Tris, 0.01% bovine serum albumin, pH 7.7) and then filtered through Whatman GF/B filters using a Brandel cell harvester. Filters were rinsed twice with 4 ml ice-cold Tris/BSA buffer and placed in 12 x 75 tubes for gamma counting. Data from platelet binding assays were transformed by Scatchard analysis (EBDALIGAND software, Elsevier-Biosoft, UK) to determine density (Bmo~)and dissociation constant (Ko) values.
5-HT Uptake Sites (JH-Paroxetine Binding Assay) Platelet suspensions were prepared as above and the protein concentrations determined using the BioRad Assay. Specific binding of 3H-paroxetine (NEN-Dupont) was determined at six-ligand concentrations (0.05-1.0 nmol/L) in the presence and absence of 1 Ixmol/L fluoxetine (Eli Lilly and Co.). The incubation mixtures consisted of platelet suspension (containing 20 ~g protein), 10 pol incubation buffer or fluoxetine, 10 ~xl 3H-paroxetine and incubation buffer to bring the total volume to I ml. Incubations were done in 12 x 75 mm polypropylene tubes. After incubation for 20 rain at 37 ° C, the samples are rapidly filtered through prewetted Whatman GF/B filters. Filters were rinsed and counted and the data analyzed, as described previously.
Table 1. S a m p l e Platelet Characteristics Depressed subjects (n = 35) (± SEM)
Controls (n = 14) (± SEM)
934.81 (64.22)
1175.75 (122.13)
5-HT uptake density standardized residuals
-.21 (. 15)
.51 (.30)
5-HT 2 density 0Cmoles/mg protein)
40.43 (4.39)
26.59 (4.27)
5-HT2 density standardized residuals
.12 (. 18)
-.30 (. 19)
0.09 a
5 -HT uptake affinity (nmol/L)
.65 (.06)
.24 (.02)
0.0001 b
5-HT uptake affinity standardized residuals
.31 (. 17)
-.77 (.27)
0.0003 ~
5-HTz affinity (nmol/L)
.70 (.08)
.40 (.06)
0.04 b
5-HTz "affinity standardized residuals
-.02 (. 19)
.05 (. 19)
0.83
Parameters 5 -HT uptake density (fmoles/mg protein)
P value 0.03 a 0.01 ° 0.04 °
~One-tailedsignificance test. OTwo-tailedsignificancetest,
ized 5 - H T 2 receptor affinity residual values. The overall significance of the model to produce standardized 5-HT uptake site density residuals was r = 0.4; F = 2.5; p = 0.06 and included following independent variables: age, 5-HT uptake site affinity, 5-HT2 receptor density, and 5-HT2 affinity. The overall significance of the model to produce standardized 5-HT 2receptor density residuals was r = 0.8; F= 17; p = 0.0001 and included the following independent variables: age, 5-HT uptake site affinity, 5-HT2 receptor density, and 5-HT2 affinity. The significance of the model to produce standardized 5-HT2 affinity values was r = 0.8, F = 19.7, p = 0.0001. The model to produce standardized 5-HT uptake affinity values was not significant (p = 0.52); and included the following independent variables: age, 5-HT uptake site affinity, 5-HT2 receptor density, and 5-HT2 affinity.
Data Analysis
Results
Between group comparisons were made using independent sample t-tests. P-values shown are uncorrected for multiple tests. Bivariate correlations between platelet measures and clinical measures were estimated using linear regression. Multiple regression analyses were used to obtain standardized residual values for 5-HT uptake site density and 5 - H T 2 receptor density in order to compare subject groups and to examine correlations between 5-HT uptake density and 5-HT2 receptor density values and symptom superfactor scores independent of other related variables (e.g., age, 5-HT uptake and 5-HT2 receptor affinity values). Multiple regression analyses were also used to derive standardized 5-HT receptor uptake affinity residual values and standard-
Table 1 summarizes patient and control sample platelet characteristics. Statistically significant differences in the predicted directions were obtained between patients and controls for 5-HT uptake site density and 5-HTz receptor density values. In addition, post-hoc analyses revealed significant differences between patients and controls for 5-HT uptake affinity and 5-HT2 receptor affinity values. Table 2 indicates F-values for bivariate linear regression analyses used to test for correlations between HRSD superfactor scores and both raw and residualized platelet measures. Figure 1 illustrates the direct, significant correlation between 5-HT uptake site density residual values and cognitive symptom superfactor scores. The regression of 5 - H T 2
Platelet Serotonin Markers and Depressive Symptomatology
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Table 2. Prediction of HRSD Superfactors Cognitive Superfactor VegetativeSuperfactor (F Values) (F Values)
Parameters 5-HT uptake site density 5-HT uptake site density residualizedvalues 5-HT2receptor density 5-HT2 receptor density residualizedvalues 5-HT uptake site affinity 5-HT uptake site affinity residualizedvalues 5-HTz receptor affinity 5-HTzreceptor affinity residualizedvalues
2.4
0. I 1
5.1" 0.002
0.38 0.36
2.6 0.128
0.42 0.49
0.66 0.45
1.14 1.27
2.9
1.3
nitive and vegetative symptom superfactors demonstrated a significant bivariate relationship (r = 0.49; p = 0.003), suggesting approximately 25% shared variance.
Discussion
"~r= 0.4, p = 0 . 0 3 .
receptor density residual values against cognitive symptom superfactor scores did not achieve statistical significance (p = 0.12). There were no statistically significant relationships between raw or standardized residualized values and vegetative superfactor scores, nor with total HRSD scores. Cog-
The data suggest that lower platelet 5-HT uptake site density values are related to increasing severity of the cognitive symptoms of depression. A trend toward a correlation between higher 5-HT2 uptake site density values and increasing severity of cognitive symptoms may also exist. In contrast, neither platelet measure showed a relationship with the severity of the vegetative symptoms of depression. W e also found no relationship between total HRSD scores and 5-HT uptake site density or 5-HT2 receptor density values, suggesting that only some dimensions of depression are predicted by platelet markers such as 5-HT uptake site density. Such relationships may be obscured by other elements of psychopathology included in the total HRSD (e.g., vegetative symptoms). Our results are consistent with the literature that implicates 5-HT abnormalities in depression, both
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Standardized 5-HT Uptake Site Density Residuals Figure I. Standardized 5-HT uptake site density residual values correlate with cognitive symptoms of depression on the HRSD. r = 0.40; F = 5.1;p =0.03.
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in differentiating depressed patients from normals, and in finding serotonergic abnormalities to be specifically associated with the severity of a "cognitive" symptom cluster (Apter et al, 1990). A large literature has demonstrated differences in platelet serotonin uptake site density values between depressed patients and normal controls. Although early work differed methodologically in using imipramine binding, most of the studies found depressed patients to have significantly lower platelet 5-HT uptake site density values than normal controls (Bunney et al 1986). In addition to serving as a state marker, platelet uptake density has been found to remain decreased in some studies during remission, suggesting value as a trait marker (reviewed in Bunney et al 1986; Healy et al 1990). A twin study by Meltzer and Arora (1988), showing higher correlations in platelet Vmaxin monozygotic versus dizygotic twins also supports the idea that platelet 5-HT uptake site characteristics may be trait markers determined by genetic variables. [3HI Imipramine labels a site closely linked to the 5-HT transporter (Mellerup and Plenge 1986) but may also label other populations of displaceable binding sites in brain and platelets. Therefore more recent studies have measured 5-HT uptake site in brain tissue and platelets using labels for the 5-HT transporter, which are more specific than imipramine. Among these are [~H] citalopram and [3HI paroxetine, which appear to identify a more homogenous population of binding sites. Binding sites for selective 5-HT uptake inhibitors (e.g., paroxetine) associated with the 5-HT nerve terminals may more closely overlap the binding site for 5-HT on the transporter protein (Marcusson and Ross 1990). Our results are also consistent with the results of prior studies that have shown increased platelet 5-HT2 receptor densities in depressed patients relative to normal controls (Biegon et al 1987, 1990a; Pandey et al 1990; Arora and Meltzer 1989). These investigators failed to find a correlation between 5-HT2 receptor density values and total HRSD or brief psychiatric rating scales (BPRS) Lyerly and Abbott 1966) scores, however. We also found no relation between total HRSD scores and 5-HT2 receptor density values, although we found a trend toward a correlation between 5-HT2 receptor density values and cognitive superfactor scores. Our finding of differences between depressed patients and controls for platelet 5-HT uptake site affinity and 5-HT2 receptor affinity values was unexpected. The unwanted
presence of antidepressant drug in the assays is an unlikely though possible explanation for this finding. The comparison was made at baseline, prior to starting on antidepressant medication, and after a week of placebo wash-out and the report of at least 2 prior weeks off all psychotropic medication. All patients received a drug screen on admission. There still could have been an effect of residual drug from distant and undocumented antidepressant drug treatment, however, in particular fluoxetine. Other explanations for this finding, besides residual drug effects, include differences in the nature (e.g., ligand binding properties) of receptor populations between depressed patients and controls, or the unusual presence of more than one receptor population in depressed patients (Bennett and Yamamura 1985). In any case, differences in Ko between depressed subjects and normals cannot explain findings related to residualized 5-HT uptake site density values obtained after removing the variance shared with Ko values. The findings of this study are consistent with other studies that indicate a specific relationship between serotonergic abnormalities and particular dimensions of mood disturbance. We previously demonstrated a specific correlation between lower CSF 5-HIAA concentrations and the severity of cognitive symptoms but not vegetative symptoms (Faustmann et al 1990). Other work also supports the specificity of the relationship between lower CSF 5-HIAA concentrations and cognitive symptoms of depression, including suicidal urges and behavior (Traskman et al 1981; ,~sberg et al 1984; Roy et al 1989). Our current findings lend support to the notion that platelet serotonin markers may be altered in parallel to central serotonin markers, such as CSF 5-HIAA, in patients with depression. An important question is whether delineating properties of the platelet 5-HT transporter and 5-HT2 receptor has any relevance for corresponding brain proteins, or whether platelet and brain 5-HT markers are unrelated, as has been previously suggested (Moret and Briley 1991). Recent work using cross-species cloning has shown that the human platelet 5-HT transporter and the brain 5-HT transporter are encoded by the same gene (Lesch et al 1993). Thus, our findings in human platelets may have relevance for understanding at least some properties of corresponding brain serotonin receptors. For example, if a functional abnormality of the 5-HT transporter protein is the direct result of a change in primary structure, it should be measurable in platelets as well as in brain.
References Apter A, van Praag HM, Plutchik R, Sevy S, Korn M, Brown SL (1990): Interrelationships among anxiety, aggression, impulsivity and mood: A serotonergically linked cluster? Psvchiato' Res 32:191-199.
Arora RC, Meltzer HY (1989): Increased serotonin 2 (5-HTz) receptor binding as measured by 3H-lysergic acid diethylamide (3H-LSD) in the blood platelets of depressed patients. Life Sci 44:725-734.
Platelet Serotonin Markers and Depressive Symptomatology
Asberg M, Bertilsson L, Martensson B, Scalia-Tomba GP, Thoren P, Traskman-Bendz L (1984): CSF monamine metabolites in melancholia. Acta Psychatr Scand 69:201-219. Bennett JP, Yamamura HI (1985): Neurotransmitter, hormone, and drug receptor binding methods. In Yamamura HI, (ed), Neurotransmitter Receptor Binding. New York: Raven Press, pp 61-89. Biegon A, Weigman A, Karp L, Ram A, Tiana S, WolffM (1987): Serotonin 5-HT2 receptor binding in blood platelets--A peripheral marker for depression? Life Sci 42:2485-2492. Biegon A, Grinspoon A, B[umenfeld B, Bleich A, ApterA, Mester R (1990a): Increased serotonin 5 - H T 2 receptor binding on blood platelets of suicidal men. Psychopharmacology 100:165-167. Biegon A, Essar N, Israeli M, Elizur A, Bruch S, Bar-Nathan A (1990b): Serotonin 5-HTz receptor binding on blood platelets as a state dependent marker in major affeetive disorder. Psychopharmacology 102:73-75. Bunney WE, Garland-Bunney B, Patel SB (1986): Biological markers in depression. Psychopathology 19(2):72-78. Coppen A, Swade C, Loudon JB (1978): Platelet 5-hydroxytryptamine accumulation in depressive illness. Clin Chim Acta 87:165-168. Csernansky JG, Sheline YI (1993): Abnormalities of Serotonin Metabolism and Nonpsychotic Psychiatric Disorders. Ann Clin Psychiatry 57:275-282. Faustmann WO, Faull KF, Whiteford HA, Borchert C, Csernansky JG (1990): CSF 5 HIAA, serum cortisol and age differentially predict vegetative and cognitive symptoms in depression. Biol Psychiato' 27:311-318. Freeman AM, Stankovic SMI, Bradley RJ, Zhang GZ, Libb JW, Nemeroff CB (1993): Tritiated platelet imipramine binding and treatment response in depressed outpatients. Depression 1:20-23. Hamilton M (1960): A rating scale for depression. J Neurol Neurosurg Psychiatry 23:56-62. Healy D, Theodorou AE, Whitehouse AM, et al (1990): [3H]-imipramine binding to previously frozen platelet membranes from depressed patients, before and after treatment Br J Psychiat O' 157:208-2 l 5. Lesch K-P, Wolozin BL, Murphy DL, Riederer (1993): Primary structure of the human platelet serotonin uptake site: Identify with the brain serotonin transporter. J Neurochem 60:2319-2322.
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Lyerly SD, Abbott PS (1966): Handbook of Psychiatric Rating Scales (1950-1964), Public Health Service Publication No. 1495. Washington, DC: Department of Health, Education, and Welfare. Marcusson JO, Ross SB ( 1990): Binding of some antidepressants to the 5-hydroxytryptamine transporter in brain and platelets. Psychopharmacology 102:145-155. Mellerup ET, Plenge P (1986): High affinity binding of 3H-imipramine to rat neuronal membranes. Psychopharmacology 89:436-439. Meltzer HY, Arora RC (1988): Genetic control of serotonin uptake in blood platelets: A twin study. Psychiatry Res 24:263-269. Meltzer HY, Lowy MT (1987): The serotonin hypothesis of depression. In: Meltzer HY, (ed), Psychopharmacology, The Third Generation of Progress. New York: Raven Press, pp 513-526. Moret C, Briley M (1991): Platelet 3H-Paroxetine Binding to the serotonin transporter is insensitive to changes in central serotonergic innervation in the rat. Psychiatry Res 38:97-104. Pandey GN, Pandey SC, Janicak PG, Merks RC, Davis JM ( 1990): Platelet serotonin-2 receptor binding sites in depression and suicide. Biol Psychiatry 28:215-222. Paul SM, Rehavi M, Skolnick P, Ballenger JC, Goodwin FK (1981): Depressed patients have decreased binding of tritiated imipramine to platelet serotonin transporter. Arch Gen Psychiatry 38:1315-1317. Rhoades HM, Overall JE (1983): The Hamilton Depression Scale: Factor sorting and profile classification. Psychopharmacology 19:91-96. Roy A, DeJong J Linnoila M (1989): Cerebrospinal fluid monoamine metabolites and suicidal behavior in depressed patients. Arch Gen Psychiatry 46:609-612. Scott M, Reading HW, Loudon JB (1979): Studies on human blood platelets in affective disorder. Psychopharmacology 60:131-135. Traskman L, Asberg M, Bertillson L, Sjostrand MO ( 1981 ): Monoamine metabolites in CSF and suicidal behavior. Arch Gen Psychiatry 38:631-636. Wagner A, Abert-Wistedt A, Asberg M, et al (1987): Effects of antidepressant treatments on platelet tritiated imipramine binding in major depressive disorder. Arch Gen Psychiato' 44:870-877.
Platelet Serotonin Markers and Depressive Symptomatology Yvette I. Sheline, Mark E. Bardgett, Jamie L. Jackson, John W. Newcomer, and John G. Csernansky
Dysfunction of brain serotonergic symptoms may be a factor in the mood and behavioral disturbances associated with depression. Platelet serotonin measures represent indirect but easily obtainable indices of brain serotonin function. To examine the specificity, of relationships between cognitive and vegetative symptom groupings and platelet serotonin measures, we assessed 35 depressed outpatients using the Hamilton Rating Scale for Depression and collected platelets after a minimum 3-week drug-free period. Platelets were also collected from 14 controls. The results showed that depressed patients had lower platelet serotonin (5-HT) uptake site density values than controls and that 5-HT uptake site density values were inversely correlated with the severity, of cognitive symptoms of depression. Platelet 5-HT2 receptor density values were higher in depressed patients than controls, and there was a trend toward a direct correlation between the cognitive symptoms of depression and 5-HTz receptor density' values. Neither platelet measure showed any relationship with the severity of the vegetative symptoms of depression. Key Words: Depression, serotonin, platelets, 5-HT uptake,
Introduction There is growing evidence that dysfunction of serotonergic systems may be an underlying factor in the pathophysiology of mood disorders (Meltzer and Lowy 1987). Among the more direct measures of central nervous system (CNS) serotonergic function is cerebrospinal fluid (CSF) concentration of 5-hydroxyindoleacetic acid (5-HIAA) concentrations, the principal metabolite of serotonin (5-HT). Depressed patients with melancholia have lower concentrations of CSF 5-HIAA than normal controls, and depressed From the Department of Psychiatry, Washington University School of Medicine, St. Louis, M163110. Address reprint requests to Yvette Sheline. MD, Assistant Professor of Psychiatry,, Department of Psychiatry, Washington University School of Medicine, 4940 Children's Place, St. Louis, MO 63110-1093. Received March 11, 1994; revised June 1, 1994.
© 1995 Society, of Biological Psychiatry
5 - H T 2 receptor
patients who make suicide attempts have lower CSF 5HIAA concentrations than other depressed patients (Asberg et al 1984). Further, increases in CSF 5-HIAA have been found after recovery from depression (Traskman et al 1981). In one follow-up study, patients with lower CSF 5-HIAA concentrations during an episode of depression were more likely to re-attempt suicide within 5 years (Roy et al 1989). Platelet 5-HT uptake sites and 5-HT2 receptors may provide indirect, but readily accessible, measures of serotonin activity. Previous studies have demonstrated decreases in platelet 5-HT uptake site densities and increases in platelet 5-HT2 receptor densities in unmedicated, depressed patients compared to normals (Paul et al 1981; Biegon et al 1987). Some investigators have reported the density of uptake sites to remain decreased after recovery in unipolar depressed 0006-3223195/$09.50 SSDI 0006-3223(94)00160-5
Platelet Serotonin Markers and Depressive Symptomatology
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1995 ;37:442447
patients (Coppen et al 1978; Scott et al 1979; Healy et al 1990). Others, however, have found that both 5-HT uptake site density decreases and 5-HTz receptor density increases associated with depression tend to normalize after treatment with antidepressants (Wagner et al 1987; Freeman et al 1993; Biegon et al 1990b). Although there may be differences in platelet 5-HT uptake site and 5 HT2 receptor density values between depressed patients and controls, it remains unknown to what extent these alterations underlie specific dimensions of mood and behavioral pathology in depression (Apter et al 1990). For this reason, we decided to examine correlations between platelet serotonin markers and the two major groupings of depressive symptoms, "cognitive" and "vegetative" symptoms. Prior factor analytic studies (Rhodes and Overall 1983) have identified cognitive and vegetative superfactors composed of items in the Hamilton Rating Scale for Depression (HRSD) (Hamilton 1960). Cognitive symptoms denote the items in the (HRSD) that affect mental functioning, such as mood and guilt, whereas vegetative symptoms denote items reflecting somatic functioning. Given the association between 5-HT abnormalities and anxiety, aggression, impulsivity, mood instability, and suicide in a variety of psychiatric disorders in addition to depression (Apter et al 1990; Csernansky and Sheline 1993) we hypothesized that platelet 5-HT markers would be correlated with cognitive symptoms of depression but not with vegetative symptoms. In an attempt to replicate prior work we also hypothesized that depressed patients would show lower 5-HT uptake site density values and higher 5-HT2 receptor density values compared to the control group.
Methods
PatientSample Patients were recruited through local newspaper advertisements to participate in controlled antidepressant drug trials, as well as this research protocol. All patients gave written informed consent prior to their participation. There were 13 men and 22 women who participated. Mean age was 44.1 _+ 14.5 (SD), range = 18-77. All patients were interviewed by board-certified and specially trained research psychiatrists (YIS and JGC) using a semi-structured clinical interview. All patients met DSM-III-R criteria for unipolar major depression at baseline. Exclusionary criteria included other primary DSM-III-R diagnoses, recent drug or alcohol abuse or dependence, major medical illnesses, and the use within the past 3 weeks of any drugs affecting central nervous system (CNS) function such as psychotropic drugs, calcium channel blockers, and [3 blockers. Symptoms were assessed using the 17 item HRSD (Hamilton 1960), which was administered after a 7-10 day placebo washout. Mean HRSD score was 22.9 -+
6.5 (SD). The range of values was 2-41, indicating that the symptoms of some patients were changed considerably during the placebo wash-out. Two groupings of depressive symptoms were assessed using the vegetative and cognitive HRSD superfactors identified by Rhoades and Overall (1983). The vegetative superfactor included sleep disturbance (early, middle, and late), somatic anxiety, gastrointestinal (GI) symptoms, general somatic symptoms, genital symptoms, hypochondriasis and weight loss (Items 4-6, 11-16). The mean vegetative superfactor score was 10.7 -+ 3.5 (SD), range 1-18. The cognitive superfactor included depressed mood, guilt, suicidal ideation, work/activities, retardation, agitation, psychic anxiety, and insight (Items 1-3, 7-10, 17). The mean cognitive superfactor score was 12.2 + 4.0 (SD) range 0-18.
Control Sample A sample of 14 subjects matched for age, gender, and socioeconomic status was recruited through advertisements. Mean age for the sample was 40.4 _+ 9.8 (SD), range 24-59, and included 10 women and 4 men. Control subjects were included if they had no current or previous DSM-III-R psychiatric diagnosis. In addition, control subjects were subject to the same drug exclusionary criteria as experimental subjects.
Platelet Samples Platelets were prepared according to a standard protocol as follows: 15 ml of blood were collected into vacutainers containing 0.3 ml of 7.5% ethylenediamine tetracetic acid (EDTA) (pH 7.5). Platelet-rich plasma (PRP) was obtained by centrifugation of the blood at 900 rpm for 10 min at 22 ° C in an IEC Centra-8R centrifuge. The PRP was removed using a plastic pipette, divided into three tubes, and recentrifuged at 3400 rpm for 15 min. Then the supernatant was separated from each platelet pellet. The pellet was resuspended in 2 ml of phosphate-buffered saline glucose (pH 7.2) and centrifuged again at 3400 rpm for 15 rain. After discarding the supernatant, the platelet pellets were immediately frozen at - 7 0 ° C for storage until analysis.
5-HT2 Receptors (~25I-LSDAssay) Randomly selected platelet pellets from each subject were slowly thawed and resuspended in 5 ml of 5 mmol/L Tris containing 0.1% EDTA (pH 7.5) and homogenized using a teflon pestle and glass homogenization tube. The homogenate was then centrifuged at 50,000 g for 15 min at 4 ° C. Each pellet was again re-suspended in 2.5 ml ice-cold incubation buffer (50 mmol/L Tris, 120 mmol/L NaCI, 5 mmol/L KC1, 2mmol/L MgC1, 0.05% ascorbic acid, pH 7.3) and rehomogenized.
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Protein concentrations were determined using the BioRad Protein Assay using bovine gamma globulin as the standard. Specific binding of lzSI-LSD (NEN Dupont) was determined at six-ligand concentrations (0.06-1.2 riM) in the presence and absence of 1 Ixmol/L spiperone (Research Biochemicals, Inc.). This method was developed in a series of initial exploratory studies to determine the range and number of ligand concentrations necessary to obtain highly reliable linear Scatchard transformations with a minimum of sample. Incubations were performed in microcentrifuge tubes and contained 80 txl of platelet suspension (10 p~g protein), 10 Ixl incubation buffer or spiperone and 10 ixl 12SI-LSD. Tubes were incubated at 37 ° C for 2 hr. The incubations were stopped by the addition of 1 ml ice-cold 4 ° C Tris/BSA buffer (50 mmol/L Tris, 0.01% bovine serum albumin, pH 7.7) and then filtered through Whatman GF/B filters using a Brandel cell harvester. Filters were rinsed twice with 4 ml ice-cold Tris/BSA buffer and placed in 12 x 75 tubes for gamma counting. Data from platelet binding assays were transformed by Scatchard analysis (EBDALIGAND software, Elsevier-Biosoft, UK) to determine density (Bmo~)and dissociation constant (Ko) values.
5-HT Uptake Sites (JH-Paroxetine Binding Assay) Platelet suspensions were prepared as above and the protein concentrations determined using the BioRad Assay. Specific binding of 3H-paroxetine (NEN-Dupont) was determined at six-ligand concentrations (0.05-1.0 nmol/L) in the presence and absence of 1 Ixmol/L fluoxetine (Eli Lilly and Co.). The incubation mixtures consisted of platelet suspension (containing 20 ~g protein), 10 pol incubation buffer or fluoxetine, 10 ~xl 3H-paroxetine and incubation buffer to bring the total volume to I ml. Incubations were done in 12 x 75 mm polypropylene tubes. After incubation for 20 rain at 37 ° C, the samples are rapidly filtered through prewetted Whatman GF/B filters. Filters were rinsed and counted and the data analyzed, as described previously.
Table 1. S a m p l e Platelet Characteristics Depressed subjects (n = 35) (± SEM)
Controls (n = 14) (± SEM)
934.81 (64.22)
1175.75 (122.13)
5-HT uptake density standardized residuals
-.21 (. 15)
.51 (.30)
5-HT 2 density 0Cmoles/mg protein)
40.43 (4.39)
26.59 (4.27)
5-HT2 density standardized residuals
.12 (. 18)
-.30 (. 19)
0.09 a
5 -HT uptake affinity (nmol/L)
.65 (.06)
.24 (.02)
0.0001 b
5-HT uptake affinity standardized residuals
.31 (. 17)
-.77 (.27)
0.0003 ~
5-HTz affinity (nmol/L)
.70 (.08)
.40 (.06)
0.04 b
5-HTz "affinity standardized residuals
-.02 (. 19)
.05 (. 19)
0.83
Parameters 5 -HT uptake density (fmoles/mg protein)
P value 0.03 a 0.01 ° 0.04 °
~One-tailedsignificance test. OTwo-tailedsignificancetest,
ized 5 - H T 2 receptor affinity residual values. The overall significance of the model to produce standardized 5-HT uptake site density residuals was r = 0.4; F = 2.5; p = 0.06 and included following independent variables: age, 5-HT uptake site affinity, 5-HT2 receptor density, and 5-HT2 affinity. The overall significance of the model to produce standardized 5-HT 2receptor density residuals was r = 0.8; F= 17; p = 0.0001 and included the following independent variables: age, 5-HT uptake site affinity, 5-HT2 receptor density, and 5-HT2 affinity. The significance of the model to produce standardized 5-HT2 affinity values was r = 0.8, F = 19.7, p = 0.0001. The model to produce standardized 5-HT uptake affinity values was not significant (p = 0.52); and included the following independent variables: age, 5-HT uptake site affinity, 5-HT2 receptor density, and 5-HT2 affinity.
Data Analysis
Results
Between group comparisons were made using independent sample t-tests. P-values shown are uncorrected for multiple tests. Bivariate correlations between platelet measures and clinical measures were estimated using linear regression. Multiple regression analyses were used to obtain standardized residual values for 5-HT uptake site density and 5 - H T 2 receptor density in order to compare subject groups and to examine correlations between 5-HT uptake density and 5-HT2 receptor density values and symptom superfactor scores independent of other related variables (e.g., age, 5-HT uptake and 5-HT2 receptor affinity values). Multiple regression analyses were also used to derive standardized 5-HT receptor uptake affinity residual values and standard-
Table 1 summarizes patient and control sample platelet characteristics. Statistically significant differences in the predicted directions were obtained between patients and controls for 5-HT uptake site density and 5-HTz receptor density values. In addition, post-hoc analyses revealed significant differences between patients and controls for 5-HT uptake affinity and 5-HT2 receptor affinity values. Table 2 indicates F-values for bivariate linear regression analyses used to test for correlations between HRSD superfactor scores and both raw and residualized platelet measures. Figure 1 illustrates the direct, significant correlation between 5-HT uptake site density residual values and cognitive symptom superfactor scores. The regression of 5 - H T 2
Platelet Serotonin Markers and Depressive Symptomatology
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Table 2. Prediction of HRSD Superfactors Cognitive Superfactor VegetativeSuperfactor (F Values) (F Values)
Parameters 5-HT uptake site density 5-HT uptake site density residualizedvalues 5-HT2receptor density 5-HT2 receptor density residualizedvalues 5-HT uptake site affinity 5-HT uptake site affinity residualizedvalues 5-HTz receptor affinity 5-HTzreceptor affinity residualizedvalues
2.4
0. I 1
5.1" 0.002
0.38 0.36
2.6 0.128
0.42 0.49
0.66 0.45
1.14 1.27
2.9
1.3
nitive and vegetative symptom superfactors demonstrated a significant bivariate relationship (r = 0.49; p = 0.003), suggesting approximately 25% shared variance.
Discussion
"~r= 0.4, p = 0 . 0 3 .
receptor density residual values against cognitive symptom superfactor scores did not achieve statistical significance (p = 0.12). There were no statistically significant relationships between raw or standardized residualized values and vegetative superfactor scores, nor with total HRSD scores. Cog-
The data suggest that lower platelet 5-HT uptake site density values are related to increasing severity of the cognitive symptoms of depression. A trend toward a correlation between higher 5-HT2 uptake site density values and increasing severity of cognitive symptoms may also exist. In contrast, neither platelet measure showed a relationship with the severity of the vegetative symptoms of depression. W e also found no relationship between total HRSD scores and 5-HT uptake site density or 5-HT2 receptor density values, suggesting that only some dimensions of depression are predicted by platelet markers such as 5-HT uptake site density. Such relationships may be obscured by other elements of psychopathology included in the total HRSD (e.g., vegetative symptoms). Our results are consistent with the literature that implicates 5-HT abnormalities in depression, both
20 19 18
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A
4
3 2 1 0
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-2
-1.5
i
!
!
i
!
I
-1
-.5
0
.5
1
1.5
2
Standardized 5-HT Uptake Site Density Residuals Figure I. Standardized 5-HT uptake site density residual values correlate with cognitive symptoms of depression on the HRSD. r = 0.40; F = 5.1;p =0.03.
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in differentiating depressed patients from normals, and in finding serotonergic abnormalities to be specifically associated with the severity of a "cognitive" symptom cluster (Apter et al, 1990). A large literature has demonstrated differences in platelet serotonin uptake site density values between depressed patients and normal controls. Although early work differed methodologically in using imipramine binding, most of the studies found depressed patients to have significantly lower platelet 5-HT uptake site density values than normal controls (Bunney et al 1986). In addition to serving as a state marker, platelet uptake density has been found to remain decreased in some studies during remission, suggesting value as a trait marker (reviewed in Bunney et al 1986; Healy et al 1990). A twin study by Meltzer and Arora (1988), showing higher correlations in platelet Vmaxin monozygotic versus dizygotic twins also supports the idea that platelet 5-HT uptake site characteristics may be trait markers determined by genetic variables. [3HI Imipramine labels a site closely linked to the 5-HT transporter (Mellerup and Plenge 1986) but may also label other populations of displaceable binding sites in brain and platelets. Therefore more recent studies have measured 5-HT uptake site in brain tissue and platelets using labels for the 5-HT transporter, which are more specific than imipramine. Among these are [~H] citalopram and [3HI paroxetine, which appear to identify a more homogenous population of binding sites. Binding sites for selective 5-HT uptake inhibitors (e.g., paroxetine) associated with the 5-HT nerve terminals may more closely overlap the binding site for 5-HT on the transporter protein (Marcusson and Ross 1990). Our results are also consistent with the results of prior studies that have shown increased platelet 5-HT2 receptor densities in depressed patients relative to normal controls (Biegon et al 1987, 1990a; Pandey et al 1990; Arora and Meltzer 1989). These investigators failed to find a correlation between 5-HT2 receptor density values and total HRSD or brief psychiatric rating scales (BPRS) Lyerly and Abbott 1966) scores, however. We also found no relation between total HRSD scores and 5-HT2 receptor density values, although we found a trend toward a correlation between 5-HT2 receptor density values and cognitive superfactor scores. Our finding of differences between depressed patients and controls for platelet 5-HT uptake site affinity and 5-HT2 receptor affinity values was unexpected. The unwanted
presence of antidepressant drug in the assays is an unlikely though possible explanation for this finding. The comparison was made at baseline, prior to starting on antidepressant medication, and after a week of placebo wash-out and the report of at least 2 prior weeks off all psychotropic medication. All patients received a drug screen on admission. There still could have been an effect of residual drug from distant and undocumented antidepressant drug treatment, however, in particular fluoxetine. Other explanations for this finding, besides residual drug effects, include differences in the nature (e.g., ligand binding properties) of receptor populations between depressed patients and controls, or the unusual presence of more than one receptor population in depressed patients (Bennett and Yamamura 1985). In any case, differences in Ko between depressed subjects and normals cannot explain findings related to residualized 5-HT uptake site density values obtained after removing the variance shared with Ko values. The findings of this study are consistent with other studies that indicate a specific relationship between serotonergic abnormalities and particular dimensions of mood disturbance. We previously demonstrated a specific correlation between lower CSF 5-HIAA concentrations and the severity of cognitive symptoms but not vegetative symptoms (Faustmann et al 1990). Other work also supports the specificity of the relationship between lower CSF 5-HIAA concentrations and cognitive symptoms of depression, including suicidal urges and behavior (Traskman et al 1981; ,~sberg et al 1984; Roy et al 1989). Our current findings lend support to the notion that platelet serotonin markers may be altered in parallel to central serotonin markers, such as CSF 5-HIAA, in patients with depression. An important question is whether delineating properties of the platelet 5-HT transporter and 5-HT2 receptor has any relevance for corresponding brain proteins, or whether platelet and brain 5-HT markers are unrelated, as has been previously suggested (Moret and Briley 1991). Recent work using cross-species cloning has shown that the human platelet 5-HT transporter and the brain 5-HT transporter are encoded by the same gene (Lesch et al 1993). Thus, our findings in human platelets may have relevance for understanding at least some properties of corresponding brain serotonin receptors. For example, if a functional abnormality of the 5-HT transporter protein is the direct result of a change in primary structure, it should be measurable in platelets as well as in brain.
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Platelet Serotonin Markers and Depressive Symptomatology
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