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Low platelet-poor plasma concentrations of serotonin in patients with combat-related posttraumatic stress disorder
Low Platelet-Poor Plasma Concentrations of Serotonin in Patients with Combat-Related Posttraumatic Stress Disorder Baruch Spivak, Yaffa Vered, Eran Graff, Ilana Blum, Roberto Mester, and Abraham Weizman Background: Combat-related posttraumatic stress disorder (CR-PTSD) is associated with a dysregulation of various neurotransmitter systems. Methods: We assessed levels of platelet-poor plasma (PPP) norepinephrine (NE), and serotonin (5-HT), and 24-hour urinary excretion of NE, dopamine (DA), and homovanillic acid (HVA) in 17 male outpatients with untreated chronic CR-PTSD (age, 33.1 6 7.4 years) and 10 normal control subjects (age, 35.8 6 2.7 years). Results: Compared with the control subjects, the PTSD patients showed significantly lower PPP 5-HT levels, elevated PPP NE levels, and significantly higher mean 24-hour urinary excretion of all three catecholamines (NE, DA, and HVA). The 24-hour urinary HVA values of the CR-PTSD patients correlated significantly and positively with the total Impact of Event Scale scores and the avoidance symptoms cluster scores, and the PPP 5-HT levels correlated negatively with the Hamilton Anxiety Rating Scale scores. The PPP NE/5-HT ratio was significantly higher in the study group than in the control subjects. Conclusions: We believe this combined enhanced noradrenergic activity and diminished 5-HT activity may be relevant to the neurobiology of CR-PTSD. Biol Psychiatry 1999;45:840 – 845 © 1999 Society of Biological Psychiatry Key Words: Posttraumatic stress disorder, serotonin, catecholamines
From the Research Unit, Ness Ziona Mental Health Center, Ness Ziona, Israel (BS, RM); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (BS, YV, EG, IB, RM, AW); Laboratory of Clinical Biochemistry, Sourasky Medical Center, Tel Aviv, Israel (YV, EG, IB); Department of Endocrinology, Rabin Medical Center, Petah Tiqva, Israel (IB); Laboratory of Biological Psychiatry, Felsenstein Medical Research Center, Beilinson Campus, Petah Tiqva, Israel (AW); and Research Unit, Geha Psychiatric Hospital, Rabin Medical Center, Petah Tiqva, Israel (AW). Address reprint requests to Baruch Spivak, MD, Ness Ziona Mental Health Center, PO Box 1, Ness Ziona 74100, Israel. Received December 1, 1997; revised June 8, 1998; accepted June 12, 1998.
© 1999 Society of Biological Psychiatry
Introduction
S
everal studies have described the simultaneous activation of the noradrenergic, dopaminergic, and serotonergic mediated systems immediately following exposure to severe psychological trauma (Charney et al 1993; Sutherland and Davidson 1994). This has been suggested to be an adaptive response necessary for survival; however, in cases of posttraumatic stress disorder (PTSD), it may become maladaptive and may lead to a chronic neurobiological dysfunction (Charney et al 1993; Southwick et al 1994). Although PTSD-related activation of the noradrenergic system has been well demonstrated, the mechanism of involvement of the other neurotransmitter systems (e.g., serotonergic and dopaminergic systems) that may be also important in the development and expression of the clinical symptoms of PTSD are less clear (Yehuda et al 1992; Southwick et al 1993; Sutherland and Davidson 1994; Weizman et al 1996; Wang et al 1997). The elevated urinary dopamine (DA), norepinephrine (NE), and epinephrine excretion observed in affected patients indicated that increased catecholaminergic activity is associated with PTSD (Yehuda et al 1992). In addition, the overlap of clinical symptoms of depression, anxiety disorders, and PTSD, together with the relative efficacy of selective serotonin reuptake inhibitors in all these disorders, suggests a possible serotonergic dysregulation in PTSD (Nagy et al 1993; Sutherland and Davidson 1994; Weizman et al 1996). The aim of the present study was to examine the involvement of the serotonergic and catecholaminergic systems in chronic combat-related PTSD (CR-PTSD).
Methods and Materials Patients The study group consisted of 17 male Israeli combat veterans with a primary diagnosis of CR-PTSD. Mean (6SD) age of the group was 33.1 6 7.4 years, and mean (6SD) duration of PTSD 0006-3223/99/$19.00 PII S0006-3223(98)00231-5
Serotonin, Catecholamines, and PTSD
symptoms was 7.8 6 6.8 years. The diagnosis of PTSD was based on the DSM-III-R criteria and was established during military service; none of the subjects had previous psychiatric treatment prior to that evaluation. The patients were interviewed (by B.S.) according to the guidelines of the Structured Clinical Interview for DSM-III-R (SCID-P) (Spitzer et al 1989) and a routine clinical interview for screening physical diseases. PTSD patients with concurrent major depression (according to the DSM-III-R criteria), somatic disease, or alcohol or drug abuse, or who were receiving any kind of pharmacologic treatment were excluded. None of the patients had received antidepressants, anxiolytics, or any other psychotropic medication on a regular basis. All patients provided informed consent to participate in the study.
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metabisulfite and sodium EDTA and immediately placed on ice. Platelet-poor plasma (PPP) was prepared by centrifugation of the blood samples at 1500 g for 15 min at 4°C. The resulting PPP was divided into two test tubes and stored frozen at 220°C until analyzed. Both patients and control subjects were instructed to avoid unusual physical activity or stress starting from 24 hours prior to the urinary collection until venous blood collection was completed. During this period they were maintained on a monoamine-free diet. Assessment with the PTSD Inventory, IES, HDRS, HARS, and TAS was made at the termination of 24-hour urinary and venous blood collection.
Laboratory Methods
Sodium octyl sulfate (SOS) was purchased from Bioanalytical Systems (BAS) (West Lafayette, IN). NE,2,3-dihydroxydibenzylamine (DHBA) and serotonin (5-HT) were purchased from Sigma (St. Louis, MO). All other chemicals used were highpressure liquid chromatography (HPLC) reagents. All buffers and solutions were passed through a 0.2 mm filter before injection into the HPLC system.
Plasma NE concentrations and urinary NE and DA concentrations were determined by a high-pressure liquid chromatography– electrochemical detection (HPLC-ECD) method (Shoup and Keife 1980). Catecholamines were absorbed into acidwashed alumina at pH 8.6 directly from 2 mL plasma or from 50 mL urine to which DHBA was added as an internal standard. After being shaken with the alumina for 10 min, the residual plasma or urine was aspirated. Catecholamines were eluted from the alumina by a small volume of 0.1 mol/L of HCIO4. The eluate was injected into the HPLC system. Urinary homovanillic acid (HVA) concentrations were determined after a 1:100 dilution of the urine. The diluted urine was injected into the HPLC system. PPP 5-HT concentrations were determined by a HPLC-ECD method after protein precipitation with 50% Trichloroacetic acid (TCA) containing sodium metabisulfite and sodium EDTA (Tagari et al 1984). The supernatant was injected into the HPLC system after filtration. The HPLC system consisted of an LDC/Milton-Roy (Riviera Beach, FL) high-pressure pump, a rheodyne injector, a reversephase (RP) chromatographic column [Octadecyl Silica (ODS) column, 4 3 250 mm, 5 mm; BAS], and an electrochemical detector (BAS) equipped with a glassy carbon electrode. The applied potential was 10.64 V. The mobile phase consisted of a monochloroacetate buffer (0.15 mol/L, pH 3.0 –3.05) containing 2 mmol/L EDTA and an ion-pair [sodium-octyl sulfate (SOS); 20 mg/L]. For 5-HT and HVA determination, methanol was added to yield a final concentration of 5%. The lower detection limit was 0.2 ng/mL for 5-HT and 50 pg/mL for NE. The coefficient of variation was 5% for both assays.
Procedure
Statistical Analysis
Twenty-four-hour urine samples were collected into a container containing 0.5 g sodium metabisulfite and 0.5 g sodium edetic acid (EDTA). The urine was kept at 4°C throughout the collection. Completeness of collection was established by the determination of 24-hour creatinine excretion and urine volume. Fivemilliliter samples of the collected urine were placed in plastic test tubes and kept frozen at 220°C until analyzed. Blood samples were collected at about 8.00 AM after a 14-hour fast into test tubes containing a few grains of sodium
Two-tailed unpaired Student’s t test and Pearson’s correlation test were used as appropriate. All results are expressed as mean 6 SD.
Control Subjects Ten mentally and physically healthy male volunteers aged 35.8 6 2.7 years who consented to participate in the study were included in the comparison group. The control subjects underwent the same diagnostic procedure (by B.S.) as the patients, to rule out any psychiatric disorder.
Instruments Severity of disease (study group only) was determined by the revised PTSD Inventory – Hebrew version, based on the DSMIII-R criteria (Solomon et al 1993), and presence of intrusive versus avoidance symptoms was determined with the Impact of Events Scale (IES) (Horowitz et al 1979). Depressive symptoms (both groups) were assessed with the 21-item Hamilton Depression Rating Scale (HDRS) (Hamilton 1960), and anxiety symptoms (both groups) with the Hamilton Anxiety Rating Scale (HARS) (Hamilton 1959). Degree of alexithymia was measured by the Toronto Alexithymia Scale (TAS) (Taylor et al 1988).
Materials
Results There was no significant difference between CR-PTSD patients and the healthy control subjects in age (t 5 2.1, df 5 25, p 5 .3). Scores on the PTSD scale (study group
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Table 1. Biochemical Data (Mean 6 SD) of CR-PTSD Patients and Healthy Control Subjects
Subjects CR-PTSD patients Controls
Platelet-poor plasma NE (pg/mL)
Platelet-poor plasma 5-HT (ng/mL)
24-hour urinary NE (mg/24 h)
24-hour urinary DA (mg/24 h)
24-hour urinary HVA (mg/24 h)
435.2 6 270.4a 188.3 6 102.1
10.9 6 7.7b 28.4 6 17.8
43.7 6 27.3b 28.6 6 17.8
265.6 6 130.0b 158.5 6 81.3
7.0 6 5.1b 4.3 6 2.2
NE, norepinephrine; 5-HT, serotonin; DA, dopamine; HVA, homovanillic acid. a p , .01 vs. healthy control subjects. b p , .05 vs. healthy control subjects.
only) ranged from 22 to 40 points (mean 6 SD, 31.9 6 4.9), and the total IES score was 31.2 6 3.8; there was no significant difference in the IES score between intrusive and avoidance symptoms (17.5 6 1.9 and 13.6 6 3.4, respectively; p 5 .4). The HDRS score was significantly higher in the CR-PTSD patients (18.7 6 7.7) than in the control subjects (1.7 6 1.4)(t 5 2.1, df 5 25, p 5 .0001). A similar difference (p 5 .0001) was found in the HARS scores (12.2 6 17 and 1.0 6 1.2, respectively). The mean TAS score was also significantly higher in the CR-PTSD group (84.6 6 11.5) than in the control subjects (63.6 6 7.4) (t 5 2.1, df 5 25, p 5 .0001). Most of the patients (15 of 17) exhibited alexithymia (TAS score .74), whereas none of the control subjects did, although 7 of the 10 control subjects had a TAS score just above the cutoff level (74 . score . 62). It should be noted that TAS score $74 indicates alexithymia, score #62 indicates nonalexithymia, and a score of 63–73 is considered borderline (Taylor et al 1988). Table 1 summarizes the biochemical data of the study population. PPP 5-HT level was significantly lower in the CR-PTSD patients than in the healthy control subjects (t 5 2.2, df 5 25, p , .05), and PPP NE was significantly higher (t 5 2.1, df 5 25, p , .01). Mean 24-hour urinary excretion of the two catecholamines (NE, DA) and the DA metabolite (HVA) was significantly higher in the CR-PTSD patients than in the
Figure 1. Correlation between 24-hour urinary HVA excretion and total Impact of Events Scale scores (r 5 .45, n 5 17, p , .05).
control subjects (for NE, t 5 2.1, df 5 25, p 5 .05; for DA, t 5 2.1, df 5 25, p , .05; for HVA, t 5 2.1, df5 25, p , .05). The 24-hour urinary HVA values in the CR-PTSD patients correlated significantly with total IES score (Figure 1) and avoidance symptoms cluster score (Figure 2)(r 5 .45, n 5 17, p , .05 in the CR-PTSD patients for both). There was a negative correlation between PPP 5-HT levels and HARS scores in the CR-PTSD patients (Figure 3)(r 5 2.45, n 5 17, p , .05), i.e., the higher the HARS score, the lower the PPP 5-HT value; this was not observed in the control group (r 5 2.30, n 5 10, ns). There was also a strong association in the CR-PTSD group, but not in the control subjects, between 24-hour urinary DA and NE excretion (r 5 .77, n 5 17, p 5 .001; for control subjects: r 5 .11, n 5 10, ns). The ratio of PPP NE to PPP 5-HT (NE/5-HT) was significantly higher in the CR-PTSD patients (44.7 6 37.3 vs. 15.2 6 16.5; t 5 2.06, df 5 25, p , .05).
Discussion Our results are in agreement with previous findings of increased 24-hour urinary catecholamine excretion (NE and DA) as well as the DA metabolite (HVA) in patients with CR-PTSD compared to healthy control subjects (Yehuda et al 1992; Kosten et al 1987). Furthermore, the
Figure 2. Correlation between 24-hour urinary HVA excretion and avoidance symptoms cluster score (r 5 .45, n 5 17, p , .05).
Serotonin, Catecholamines, and PTSD
Figure 3. Correlation between PPP 5-HT values and Hamilton Anxiety Rating Scale (HAM-A) scores (r 5 2.45, n 5 17, p , .05).
urinary NE and DA measures showed a significant intercorrelation in the PTSD group but not in the control subjects. The correlation between urinary DA and HVA in the PTSD patients did not reach statistical significance, though a strong trend (p 5 .08) was detected. The lack of statistical significance may be due to the small sample size; however, only urinary HVA values correlated significantly with the severity of PTSD symptomatology (total IES scores), particularly with the avoidant symptoms cluster (Figures 1 and 2). Several investigations using peripheral measures of resting states (Davidson et al 1985; Kosten et al 1987; Lerer et al 1987; Perry et al 1987; Yehuda et al 1992) and for stressful conditions (Blanchard et al 1991; McFall et al 1990) have provided strong support for noradrenergic dysregulation in PTSD. Furthermore, several physiological and biochemical studies have shown preliminary evidence of central noradrenergic dysregulation in CR-PTSD (Reiney et al 1987; Southwick et al 1993; Bremner et al 1997), though one failed to replicate the finding of elevated urinary 24-hour NE excretion (Pitman and Orr 1990). Many of the chronic symptoms of PTSD, such as elevated anxiety level, insomnia, exaggerated startle response, and autonomic hyperarousal, may be related to increased noradrenergic tone (Charney et al 1993). By contrast, only two studies, to the best of our knowledge, have directly investigated peripheral DA levels in CR-PTSD. In one, plasma DA was shown to be significantly higher in CR-PTSD patients compared to major depressive patients and healthy control subjects (Hamner et al 1990), and in the other 24-hour urinary DA excretion was significantly higher in CR-PTSD inpatients compared to CR-PTSD outpatients and healthy control subjects (Yehuda et al 1992). Our findings are consistent with those of Yehuda et al (1992) except that we observed elevated 24-hour urinary DA excretion in CR-PTSD outpatients. This difference may be related to the relatively
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small number of outpatients (n 5 7) in the other study. In any case, this finding suggests a possible role for the dopaminergic system in this disorder. This assumption is supported by recent evidence of stress-induced hyperactivity of the central dopaminergic system (Charney et al 1993; Sutherland and Davidson 1994). Some specific PTSD symptoms, including generalized anxiety, panic attacks, hypervigilance, and exaggerated startle response, as well as memory and attention alteration, may be linked to DA system hyperactivity (Bremner et al 1993; Charney et al 1993). The increased urinary HVA excretion and its correlation with PTSD severity (total IES score) may indicate overactive DA neurotransmission in CR-PTSD. Animal and human studies suggest that pharmacology-induced modulations in brain DA turnover are reflected in parallel alterations in peripheral HVA concentrations (Kendler et al 1982; Pickar et al 1986). Although the proportion of peripheral HVA contributed by brain HVA has not been fully clarified (Pickar et al 1986), it is possible that the enhanced urinary HVA excretion in CR-PTSD may indicate augmented central DA turnover. It is of note that traumatic stress can enhance DA release and metabolism, especially in the mesoprefrontal cortical DA system (Herman et al 1982; Mantz et al 1989; Deutch and Roth 1990). The findings of low PPP 5-HT level in our study support previous observations that a dysregulation in serotonergic activity may play a significant role in the pathophysiology and clinical manifestations of PTSD. Altered 5-HT activity following severe stress or trauma has been reported in animals (Dunn 1989; Shimizu et al 1992; Moreau et al 1993; Kawahara et al 1993). Furthermore, increased central 5-HT activity is required for successful adaptation to stress and prevention of the development of learned helplessness (Joseph and Kennett 1983; Petty et al 1992, 1996). Low cerebrospinal fluid 5-hydroxyindoleacetic acid (5-HIAA) has been associated with suicide and destructive–aggressive behavior, and central serotonergic system dysfunction has been suggested as an important correlate of impulsive–aggressive behavior and impulse control (Cocarro 1989; Van Praag et al 1990). Hypofunction of the central serotonergic system has been implicated in increased startle reaction, which together with irritability and impulsive aggression, is the most disturbing symptom in chronic CR-PTSD (Marmar et al 1996). In addition, animal models have shown that successful stress adaptation is associated with increased central 5-HT function (Joseph and Kennett 1983) and that 5-HT pathways apparently have particular relevance in the development of such PTSD symptoms as conditioned avoidance behaviors (Hensman et al 1991; Sutherland and Davidson 1994). A significantly lower number of platelet 5-HT transporters, measured by [3H] paroxetine binding,
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was detected in PTSD patients as compared to normal control subjects (Arora et al 1993; Fichtner et al 1994), although a recent study failed to demonstrate alteration in [3H] imipramine binding to platelets of PTSD patients (Weizman et al 1996). Treatment of CR-PTSD patients with serotonin reuptake inhibitors has a beneficial effect on intrusion, avoidance, and arousal symptoms, and reduces anger, irritability, and explosiveness (Marmar et al 1996; Nagy et al 1993; Shay 1992). Taken together, these neurobehavioral, biochemical, and pharmacologic studies and our results suggest that serotonin deficit is an important factor in the development and persistence of PTSD symptoms. The major finding of our study was the increased PPP NE/5-HT ratio in PTSD patients as compared to healthy control subjects. Previous studies have reported the parallel involvement of several neural systems in PTSD (Charney et al 1993; Southwick et al 1994, 1997). It is possible that both NE and 5-HT systems are dysfunctional in PTSD patients. Both systems are anatomically closely connected and functionally highly interactive. Both are involved in a neuronal network hypothesized to mediate the stressinduced learned helplessness animal model (Petty et al 1996). Preclinical studies indicate that increases in 5-HT neurotransmission may reduce noradrenergic neuronal activity, e.g., stimulation of dorsal raphe inhibits locus coeruleus firing (Segal 1979). Furthermore, a clinical study demonstrated that the 5-HT reuptake inhibitor fluvoxamine blocks the anxiogenic effect of yohimbine (Goddard et al 1990). It was suggested that the beneficial effect of selective serotonin reuptake inhibitors in PTSD patients may be related not only to a direct serotonergic stimulation but also to secondary inhibitory effect on the noradrenergic system (Nagy et al 1993; Petty et al 1996). Perhaps it is the simultaneous increase in noradrenergic tone and decrease in serotonergic activity that is relevant to the pathophysiology of PTSD. This dissociation of NE and 5-HT activities, in contrast to their simultaneous decrease in major depression, may also be important to the unique alteration in hypothalamic–pituitary–adrenal axis activity reported in PTSD (Yehuda et al 1993); however, a recent study has suggested the presence of two neurobiological subgroups of PTSD patients: one with a sensitized noradrenergic system (as measured by behavioral responses to yohimbine) and the other with a sensitized serotonergic system, as assessed by behavioral responsitivity to meta-chlorophenylpiperazine (Southwick et al 1997). Nevertheless, it should be noted that we determined levels of peripheral, not central NE and 5-HT concentrations (despite our efforts to keep the subjects on a restricted diet and our use of PPP and urine collection for biochemical determination). Furthermore, it is still possible that the PTSD patients were unable to avoid unusual
physical activity or stress during the preceding urine and blood collection. In such a case, we did not obtain true NE baseline levels. A further study with a larger population of PTSD patients and assessment of NE/5-HT turnover in the cerebrospinal fluid may better reflect the central noradrenergic and serotonergic systems function in CR-PTSD.
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From the Research Unit, Ness Ziona Mental Health Center, Ness Ziona, Israel (BS, RM); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (BS, YV, EG, IB, RM, AW); Laboratory of Clinical Biochemistry, Sourasky Medical Center, Tel Aviv, Israel (YV, EG, IB); Department of Endocrinology, Rabin Medical Center, Petah Tiqva, Israel (IB); Laboratory of Biological Psychiatry, Felsenstein Medical Research Center, Beilinson Campus, Petah Tiqva, Israel (AW); and Research Unit, Geha Psychiatric Hospital, Rabin Medical Center, Petah Tiqva, Israel (AW). Address reprint requests to Baruch Spivak, MD, Ness Ziona Mental Health Center, PO Box 1, Ness Ziona 74100, Israel. Received December 1, 1997; revised June 8, 1998; accepted June 12, 1998.
© 1999 Society of Biological Psychiatry
Introduction
S
everal studies have described the simultaneous activation of the noradrenergic, dopaminergic, and serotonergic mediated systems immediately following exposure to severe psychological trauma (Charney et al 1993; Sutherland and Davidson 1994). This has been suggested to be an adaptive response necessary for survival; however, in cases of posttraumatic stress disorder (PTSD), it may become maladaptive and may lead to a chronic neurobiological dysfunction (Charney et al 1993; Southwick et al 1994). Although PTSD-related activation of the noradrenergic system has been well demonstrated, the mechanism of involvement of the other neurotransmitter systems (e.g., serotonergic and dopaminergic systems) that may be also important in the development and expression of the clinical symptoms of PTSD are less clear (Yehuda et al 1992; Southwick et al 1993; Sutherland and Davidson 1994; Weizman et al 1996; Wang et al 1997). The elevated urinary dopamine (DA), norepinephrine (NE), and epinephrine excretion observed in affected patients indicated that increased catecholaminergic activity is associated with PTSD (Yehuda et al 1992). In addition, the overlap of clinical symptoms of depression, anxiety disorders, and PTSD, together with the relative efficacy of selective serotonin reuptake inhibitors in all these disorders, suggests a possible serotonergic dysregulation in PTSD (Nagy et al 1993; Sutherland and Davidson 1994; Weizman et al 1996). The aim of the present study was to examine the involvement of the serotonergic and catecholaminergic systems in chronic combat-related PTSD (CR-PTSD).
Methods and Materials Patients The study group consisted of 17 male Israeli combat veterans with a primary diagnosis of CR-PTSD. Mean (6SD) age of the group was 33.1 6 7.4 years, and mean (6SD) duration of PTSD 0006-3223/99/$19.00 PII S0006-3223(98)00231-5
Serotonin, Catecholamines, and PTSD
symptoms was 7.8 6 6.8 years. The diagnosis of PTSD was based on the DSM-III-R criteria and was established during military service; none of the subjects had previous psychiatric treatment prior to that evaluation. The patients were interviewed (by B.S.) according to the guidelines of the Structured Clinical Interview for DSM-III-R (SCID-P) (Spitzer et al 1989) and a routine clinical interview for screening physical diseases. PTSD patients with concurrent major depression (according to the DSM-III-R criteria), somatic disease, or alcohol or drug abuse, or who were receiving any kind of pharmacologic treatment were excluded. None of the patients had received antidepressants, anxiolytics, or any other psychotropic medication on a regular basis. All patients provided informed consent to participate in the study.
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metabisulfite and sodium EDTA and immediately placed on ice. Platelet-poor plasma (PPP) was prepared by centrifugation of the blood samples at 1500 g for 15 min at 4°C. The resulting PPP was divided into two test tubes and stored frozen at 220°C until analyzed. Both patients and control subjects were instructed to avoid unusual physical activity or stress starting from 24 hours prior to the urinary collection until venous blood collection was completed. During this period they were maintained on a monoamine-free diet. Assessment with the PTSD Inventory, IES, HDRS, HARS, and TAS was made at the termination of 24-hour urinary and venous blood collection.
Laboratory Methods
Sodium octyl sulfate (SOS) was purchased from Bioanalytical Systems (BAS) (West Lafayette, IN). NE,2,3-dihydroxydibenzylamine (DHBA) and serotonin (5-HT) were purchased from Sigma (St. Louis, MO). All other chemicals used were highpressure liquid chromatography (HPLC) reagents. All buffers and solutions were passed through a 0.2 mm filter before injection into the HPLC system.
Plasma NE concentrations and urinary NE and DA concentrations were determined by a high-pressure liquid chromatography– electrochemical detection (HPLC-ECD) method (Shoup and Keife 1980). Catecholamines were absorbed into acidwashed alumina at pH 8.6 directly from 2 mL plasma or from 50 mL urine to which DHBA was added as an internal standard. After being shaken with the alumina for 10 min, the residual plasma or urine was aspirated. Catecholamines were eluted from the alumina by a small volume of 0.1 mol/L of HCIO4. The eluate was injected into the HPLC system. Urinary homovanillic acid (HVA) concentrations were determined after a 1:100 dilution of the urine. The diluted urine was injected into the HPLC system. PPP 5-HT concentrations were determined by a HPLC-ECD method after protein precipitation with 50% Trichloroacetic acid (TCA) containing sodium metabisulfite and sodium EDTA (Tagari et al 1984). The supernatant was injected into the HPLC system after filtration. The HPLC system consisted of an LDC/Milton-Roy (Riviera Beach, FL) high-pressure pump, a rheodyne injector, a reversephase (RP) chromatographic column [Octadecyl Silica (ODS) column, 4 3 250 mm, 5 mm; BAS], and an electrochemical detector (BAS) equipped with a glassy carbon electrode. The applied potential was 10.64 V. The mobile phase consisted of a monochloroacetate buffer (0.15 mol/L, pH 3.0 –3.05) containing 2 mmol/L EDTA and an ion-pair [sodium-octyl sulfate (SOS); 20 mg/L]. For 5-HT and HVA determination, methanol was added to yield a final concentration of 5%. The lower detection limit was 0.2 ng/mL for 5-HT and 50 pg/mL for NE. The coefficient of variation was 5% for both assays.
Procedure
Statistical Analysis
Twenty-four-hour urine samples were collected into a container containing 0.5 g sodium metabisulfite and 0.5 g sodium edetic acid (EDTA). The urine was kept at 4°C throughout the collection. Completeness of collection was established by the determination of 24-hour creatinine excretion and urine volume. Fivemilliliter samples of the collected urine were placed in plastic test tubes and kept frozen at 220°C until analyzed. Blood samples were collected at about 8.00 AM after a 14-hour fast into test tubes containing a few grains of sodium
Two-tailed unpaired Student’s t test and Pearson’s correlation test were used as appropriate. All results are expressed as mean 6 SD.
Control Subjects Ten mentally and physically healthy male volunteers aged 35.8 6 2.7 years who consented to participate in the study were included in the comparison group. The control subjects underwent the same diagnostic procedure (by B.S.) as the patients, to rule out any psychiatric disorder.
Instruments Severity of disease (study group only) was determined by the revised PTSD Inventory – Hebrew version, based on the DSMIII-R criteria (Solomon et al 1993), and presence of intrusive versus avoidance symptoms was determined with the Impact of Events Scale (IES) (Horowitz et al 1979). Depressive symptoms (both groups) were assessed with the 21-item Hamilton Depression Rating Scale (HDRS) (Hamilton 1960), and anxiety symptoms (both groups) with the Hamilton Anxiety Rating Scale (HARS) (Hamilton 1959). Degree of alexithymia was measured by the Toronto Alexithymia Scale (TAS) (Taylor et al 1988).
Materials
Results There was no significant difference between CR-PTSD patients and the healthy control subjects in age (t 5 2.1, df 5 25, p 5 .3). Scores on the PTSD scale (study group
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Table 1. Biochemical Data (Mean 6 SD) of CR-PTSD Patients and Healthy Control Subjects
Subjects CR-PTSD patients Controls
Platelet-poor plasma NE (pg/mL)
Platelet-poor plasma 5-HT (ng/mL)
24-hour urinary NE (mg/24 h)
24-hour urinary DA (mg/24 h)
24-hour urinary HVA (mg/24 h)
435.2 6 270.4a 188.3 6 102.1
10.9 6 7.7b 28.4 6 17.8
43.7 6 27.3b 28.6 6 17.8
265.6 6 130.0b 158.5 6 81.3
7.0 6 5.1b 4.3 6 2.2
NE, norepinephrine; 5-HT, serotonin; DA, dopamine; HVA, homovanillic acid. a p , .01 vs. healthy control subjects. b p , .05 vs. healthy control subjects.
only) ranged from 22 to 40 points (mean 6 SD, 31.9 6 4.9), and the total IES score was 31.2 6 3.8; there was no significant difference in the IES score between intrusive and avoidance symptoms (17.5 6 1.9 and 13.6 6 3.4, respectively; p 5 .4). The HDRS score was significantly higher in the CR-PTSD patients (18.7 6 7.7) than in the control subjects (1.7 6 1.4)(t 5 2.1, df 5 25, p 5 .0001). A similar difference (p 5 .0001) was found in the HARS scores (12.2 6 17 and 1.0 6 1.2, respectively). The mean TAS score was also significantly higher in the CR-PTSD group (84.6 6 11.5) than in the control subjects (63.6 6 7.4) (t 5 2.1, df 5 25, p 5 .0001). Most of the patients (15 of 17) exhibited alexithymia (TAS score .74), whereas none of the control subjects did, although 7 of the 10 control subjects had a TAS score just above the cutoff level (74 . score . 62). It should be noted that TAS score $74 indicates alexithymia, score #62 indicates nonalexithymia, and a score of 63–73 is considered borderline (Taylor et al 1988). Table 1 summarizes the biochemical data of the study population. PPP 5-HT level was significantly lower in the CR-PTSD patients than in the healthy control subjects (t 5 2.2, df 5 25, p , .05), and PPP NE was significantly higher (t 5 2.1, df 5 25, p , .01). Mean 24-hour urinary excretion of the two catecholamines (NE, DA) and the DA metabolite (HVA) was significantly higher in the CR-PTSD patients than in the
Figure 1. Correlation between 24-hour urinary HVA excretion and total Impact of Events Scale scores (r 5 .45, n 5 17, p , .05).
control subjects (for NE, t 5 2.1, df 5 25, p 5 .05; for DA, t 5 2.1, df 5 25, p , .05; for HVA, t 5 2.1, df5 25, p , .05). The 24-hour urinary HVA values in the CR-PTSD patients correlated significantly with total IES score (Figure 1) and avoidance symptoms cluster score (Figure 2)(r 5 .45, n 5 17, p , .05 in the CR-PTSD patients for both). There was a negative correlation between PPP 5-HT levels and HARS scores in the CR-PTSD patients (Figure 3)(r 5 2.45, n 5 17, p , .05), i.e., the higher the HARS score, the lower the PPP 5-HT value; this was not observed in the control group (r 5 2.30, n 5 10, ns). There was also a strong association in the CR-PTSD group, but not in the control subjects, between 24-hour urinary DA and NE excretion (r 5 .77, n 5 17, p 5 .001; for control subjects: r 5 .11, n 5 10, ns). The ratio of PPP NE to PPP 5-HT (NE/5-HT) was significantly higher in the CR-PTSD patients (44.7 6 37.3 vs. 15.2 6 16.5; t 5 2.06, df 5 25, p , .05).
Discussion Our results are in agreement with previous findings of increased 24-hour urinary catecholamine excretion (NE and DA) as well as the DA metabolite (HVA) in patients with CR-PTSD compared to healthy control subjects (Yehuda et al 1992; Kosten et al 1987). Furthermore, the
Figure 2. Correlation between 24-hour urinary HVA excretion and avoidance symptoms cluster score (r 5 .45, n 5 17, p , .05).
Serotonin, Catecholamines, and PTSD
Figure 3. Correlation between PPP 5-HT values and Hamilton Anxiety Rating Scale (HAM-A) scores (r 5 2.45, n 5 17, p , .05).
urinary NE and DA measures showed a significant intercorrelation in the PTSD group but not in the control subjects. The correlation between urinary DA and HVA in the PTSD patients did not reach statistical significance, though a strong trend (p 5 .08) was detected. The lack of statistical significance may be due to the small sample size; however, only urinary HVA values correlated significantly with the severity of PTSD symptomatology (total IES scores), particularly with the avoidant symptoms cluster (Figures 1 and 2). Several investigations using peripheral measures of resting states (Davidson et al 1985; Kosten et al 1987; Lerer et al 1987; Perry et al 1987; Yehuda et al 1992) and for stressful conditions (Blanchard et al 1991; McFall et al 1990) have provided strong support for noradrenergic dysregulation in PTSD. Furthermore, several physiological and biochemical studies have shown preliminary evidence of central noradrenergic dysregulation in CR-PTSD (Reiney et al 1987; Southwick et al 1993; Bremner et al 1997), though one failed to replicate the finding of elevated urinary 24-hour NE excretion (Pitman and Orr 1990). Many of the chronic symptoms of PTSD, such as elevated anxiety level, insomnia, exaggerated startle response, and autonomic hyperarousal, may be related to increased noradrenergic tone (Charney et al 1993). By contrast, only two studies, to the best of our knowledge, have directly investigated peripheral DA levels in CR-PTSD. In one, plasma DA was shown to be significantly higher in CR-PTSD patients compared to major depressive patients and healthy control subjects (Hamner et al 1990), and in the other 24-hour urinary DA excretion was significantly higher in CR-PTSD inpatients compared to CR-PTSD outpatients and healthy control subjects (Yehuda et al 1992). Our findings are consistent with those of Yehuda et al (1992) except that we observed elevated 24-hour urinary DA excretion in CR-PTSD outpatients. This difference may be related to the relatively
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small number of outpatients (n 5 7) in the other study. In any case, this finding suggests a possible role for the dopaminergic system in this disorder. This assumption is supported by recent evidence of stress-induced hyperactivity of the central dopaminergic system (Charney et al 1993; Sutherland and Davidson 1994). Some specific PTSD symptoms, including generalized anxiety, panic attacks, hypervigilance, and exaggerated startle response, as well as memory and attention alteration, may be linked to DA system hyperactivity (Bremner et al 1993; Charney et al 1993). The increased urinary HVA excretion and its correlation with PTSD severity (total IES score) may indicate overactive DA neurotransmission in CR-PTSD. Animal and human studies suggest that pharmacology-induced modulations in brain DA turnover are reflected in parallel alterations in peripheral HVA concentrations (Kendler et al 1982; Pickar et al 1986). Although the proportion of peripheral HVA contributed by brain HVA has not been fully clarified (Pickar et al 1986), it is possible that the enhanced urinary HVA excretion in CR-PTSD may indicate augmented central DA turnover. It is of note that traumatic stress can enhance DA release and metabolism, especially in the mesoprefrontal cortical DA system (Herman et al 1982; Mantz et al 1989; Deutch and Roth 1990). The findings of low PPP 5-HT level in our study support previous observations that a dysregulation in serotonergic activity may play a significant role in the pathophysiology and clinical manifestations of PTSD. Altered 5-HT activity following severe stress or trauma has been reported in animals (Dunn 1989; Shimizu et al 1992; Moreau et al 1993; Kawahara et al 1993). Furthermore, increased central 5-HT activity is required for successful adaptation to stress and prevention of the development of learned helplessness (Joseph and Kennett 1983; Petty et al 1992, 1996). Low cerebrospinal fluid 5-hydroxyindoleacetic acid (5-HIAA) has been associated with suicide and destructive–aggressive behavior, and central serotonergic system dysfunction has been suggested as an important correlate of impulsive–aggressive behavior and impulse control (Cocarro 1989; Van Praag et al 1990). Hypofunction of the central serotonergic system has been implicated in increased startle reaction, which together with irritability and impulsive aggression, is the most disturbing symptom in chronic CR-PTSD (Marmar et al 1996). In addition, animal models have shown that successful stress adaptation is associated with increased central 5-HT function (Joseph and Kennett 1983) and that 5-HT pathways apparently have particular relevance in the development of such PTSD symptoms as conditioned avoidance behaviors (Hensman et al 1991; Sutherland and Davidson 1994). A significantly lower number of platelet 5-HT transporters, measured by [3H] paroxetine binding,
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was detected in PTSD patients as compared to normal control subjects (Arora et al 1993; Fichtner et al 1994), although a recent study failed to demonstrate alteration in [3H] imipramine binding to platelets of PTSD patients (Weizman et al 1996). Treatment of CR-PTSD patients with serotonin reuptake inhibitors has a beneficial effect on intrusion, avoidance, and arousal symptoms, and reduces anger, irritability, and explosiveness (Marmar et al 1996; Nagy et al 1993; Shay 1992). Taken together, these neurobehavioral, biochemical, and pharmacologic studies and our results suggest that serotonin deficit is an important factor in the development and persistence of PTSD symptoms. The major finding of our study was the increased PPP NE/5-HT ratio in PTSD patients as compared to healthy control subjects. Previous studies have reported the parallel involvement of several neural systems in PTSD (Charney et al 1993; Southwick et al 1994, 1997). It is possible that both NE and 5-HT systems are dysfunctional in PTSD patients. Both systems are anatomically closely connected and functionally highly interactive. Both are involved in a neuronal network hypothesized to mediate the stressinduced learned helplessness animal model (Petty et al 1996). Preclinical studies indicate that increases in 5-HT neurotransmission may reduce noradrenergic neuronal activity, e.g., stimulation of dorsal raphe inhibits locus coeruleus firing (Segal 1979). Furthermore, a clinical study demonstrated that the 5-HT reuptake inhibitor fluvoxamine blocks the anxiogenic effect of yohimbine (Goddard et al 1990). It was suggested that the beneficial effect of selective serotonin reuptake inhibitors in PTSD patients may be related not only to a direct serotonergic stimulation but also to secondary inhibitory effect on the noradrenergic system (Nagy et al 1993; Petty et al 1996). Perhaps it is the simultaneous increase in noradrenergic tone and decrease in serotonergic activity that is relevant to the pathophysiology of PTSD. This dissociation of NE and 5-HT activities, in contrast to their simultaneous decrease in major depression, may also be important to the unique alteration in hypothalamic–pituitary–adrenal axis activity reported in PTSD (Yehuda et al 1993); however, a recent study has suggested the presence of two neurobiological subgroups of PTSD patients: one with a sensitized noradrenergic system (as measured by behavioral responses to yohimbine) and the other with a sensitized serotonergic system, as assessed by behavioral responsitivity to meta-chlorophenylpiperazine (Southwick et al 1997). Nevertheless, it should be noted that we determined levels of peripheral, not central NE and 5-HT concentrations (despite our efforts to keep the subjects on a restricted diet and our use of PPP and urine collection for biochemical determination). Furthermore, it is still possible that the PTSD patients were unable to avoid unusual
physical activity or stress during the preceding urine and blood collection. In such a case, we did not obtain true NE baseline levels. A further study with a larger population of PTSD patients and assessment of NE/5-HT turnover in the cerebrospinal fluid may better reflect the central noradrenergic and serotonergic systems function in CR-PTSD.
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