Release of PACAP-38 in episodic cluster headache patients – an exploratory study

Nine male ECH patients treated in our outpatient clinic (without other known neurological diseases) were selected in accordance with the criteria of the Headache Classification Committee of the International Headache Society. Nine age-matched male healthy volunteers screened for non-reported/non-treated headaches served as controls (age of healthy control subjects: 40.1 ± 12.0 years old, age of EHC patients: 40.3 ± 9.6 years old). Coexisting depression in patients was ruled out by Hamilton’s depression scale. A detailed questionnaire was used to assess the features of the headache disorder. The clinical and demographic data are summarized in Table 1.

The study was approved by the Ethics Committee of the Faculty of Medicine, University of Szeged (87/2009). All participants gave their written informed consent in accordance with the Declaration of Helsinki. There were no restrictions regarding food and drink intake. The study was performed between 2010 and 2012 years. Blood samples (6 ml per subject) were taken from the cubital vein in the routinely used clinical blood sampling plastic tubes containing 12 mg ethylenediaminetetraacetic acid (BD Vacutainer, Becton Dickinson Hungary Plc., Hungary) and 1200 IU peptidase inhibitor, aprotinin (Gordox, Richter Gedeon Plc., Hungary). A single blood sample was taken in supine position after a rest of 10 min from the controls, whereas samples were taken from ECH patients during the acute spontaneous attack (when they produced typical symptoms of CH [22]) and/or in the absolute attack-free, inter-bout period (out of the cluster episode—by more than a month) between 8 a.m. and 4 p.m. Samples from both periods (during attack and outside attack) could be obtained in 5 cases (it was unintentional selection), which could be used in self-controlled comparison. Patients were asked not to start their usual treatment before sampling even during the ictal sampling. Exact date and time of blood samplings are presented in the Table 2.

Blood collection, centrifugation, storage and PACAP-38 measurement were carried out with specific and sensitive radioimmunoassay (RIA). Briefly, following centrifugation of the plasma samples (2000 rpm at +4 °C for 10 min), the peptide was extracted from the plasma into three volumes of absolute alcohol. After precipitation and a second centrifugation, the samples were dried under a nitrogen flow and resuspended in 300 μl of assay buffer before RIA determination. The assay was prepared in 1 mL 0.05 mol/L (pH 7.4) phosphate buffer containing 0.1 mol/L sodium chloride, 0.25 % (w/v) bovine serum albumin and 0.05 % (w/v) sodium azide. The tracer mono-¹²⁵I-labeled PACAP24–38 (5000 cpm/tube, 124 Bq/1.72 fmol) was prepared in our laboratory. Ovine PACAP38 was used as a RIA standard ranging from 0 to 1000 fmol/mL. The PACAP-38 antiserum (88111-3, dilution 1:10000 obtained from Abcam UK), the RIA tracer and the standard or unknown samples were measured into polypropylene tubes with the assay buffer. After incubation for 48–72 h at +4 °C, the antibody-bound peptide was separated from the free peptide by the addition of separating solution. Following centrifugation (3000 rpm at +4 °C for 20 min), the contents of the tubes were gently decanted and the radioactivity of the precipitates was measured in a gamma counter (Gamma, type: NZ310). The PACAP-38 concentrations of the unknown samples were read from calibration curves.

Data are expressed as median, interquartile range (IQR), minimum and maximum or individual values on the graphs. The distributions of data populations were checked with the Shapiro-Wilk normality test and the Levene’s test for analysis of the equality of variances was also applied.