Materials and methods

1. 1. Participants

Ninety three subjects with combat related PTSD participated in the study. All participants were Croatian male war veterans, aged 28-48 years, all Caucasians, who were hospitalized at the Referral Centre for the Stress Related Disorders of the Ministry of Health of the Republic of Croatia, Regional Center for Psychotrauma, in the University Hospital Dubrava, Zagreb, Croatia, from 1999 to 2002. The diagnosis of current and chronic PTSD was conducted according to the Structured Clinical Interview based on DSM-IV(SCID). The subjects were asked to describe their traumatic experiences and were given enough time to talk about these and other psychiatric disturbances. Different clinical symptoms (trauma-related, psychotic, and depressive), occurring in this cadre of war veterans were assessed with the CAPS, the Positive and Negative Syndrome Scale (PANSS), and the Hamilton Rating Scales for Depression (HAM-D) and Anxiety (HAM-A). All patients were war veterans who had been on active duty in the Croatian armed forces (range of 1-4 years, most with 3 years of continuous combat experience), had similar social and cultural backgrounds, and the great majority were married. All were screened with a comprehensive multidisciplinary evaluation (conducted by 2 psychiatrists and a psychologist) prior to entry into inpatient treatment. Subjects were excluded from the study if they had a positive family history of psychosis, or a history of schizophrenia, schizoaffective disorder or bipolar disorder, a serious concomitant medical condition, a history of seizures or misuse of alcohol or drugs (recent use of any psychotropic drugs within one month of baseline), clinically significant abnormalities in electrocardiogram or laboratory findings, or a serious risk of suicide. Combat-related symptoms included intrusive images of screaming soldiers, fire, bombing, rocketing, etc. Individuals taking cholesterol-lowering drugs were excluded. The procedures were fully explained and written informed consent was obtained from all patients. The local Ethics committee approved this protocol.

The control group consisted of 124 healthy male volunteers, with no personal or family history of psychopathology, and receiving no medical treatment. None of the healthy subjects were receiving psychiatric or related treatment before the samples were selected. Groups were matched on age, gender, smoking, and other socio-demographic characteristics. The control subjects agreed, and provided written informed consent, to participate in the study and to provide a blood sample.

1.2. Biochemical determination

A forearm vein was cannulated for blood sampling at 08.00 a.m., after an overnight fasting. Blood samples (8 ml) were drawn in a plastic syringe with 2 ml of acid citrate dextrose anticoagulant. Platelet-rich-plasma (PRP) was obtained by centrifugation (935 x g) for 70 s at room temperature. Platelets were sedimented by further centrifugation of PRP at 10,000 x g for 5 min. The platelet pellet was washed with saline and centrifuged again. Platelet 5-HT concentration was determined by the spectrofluorimetric method, as previously described [15,16,22]. Platelet MAO activity was determined spectrofluorimetrically using kynuramine as a substrate, as previously described [16]. Platelet protein levels were measured by the method of Lowry et al. [23]. Serum lipid levels—total cholesterol, high-density lipoprotein (HDL), and triglycerides (TG) levels—were determined by enzymatic color test, while serum low-density lipoprotein (LDL) levels were measured using an enzymatic clearance assay. Serum thyroid hormones: total and free triiodothyronine (T3) levels were assayed using an luminoimmunochemical assay kit (Johnson and Johnson Clinical Diagnostic Products, Amersham, UK). Plasma DBH activity was determined by a photometric assay, using tyramine as a substrate, by the method of Nagatsu and Udenfriend [24]. Cortisol levels were determined using a commercially available radioimmunoassay kit from Diagnostic Products Cooperation, CA, USA.

1.3. Data analysis

All data (expressed as mean ± S.D.) were evaluated by one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test. The level of significance was p<0.05.

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