5 CHAPTER 5 100 temperature for 20 minutes (2,000 rpm, 622 g). The supernatant was transferred to a 15-mL polypropylene tube (Greiner Bio-One CELLSTAR®), inverted several times, and divided in 0.5mL aliquots (1.0 mL NuncTM cryotubes). Plasma samples were stored at -80°C until further use; no extra freeze-thaw cycles were allowed. Migraine-like headache and criteria Participants were notified that GTN could potentially induce a headache, without any information regarding the expected onset or course. Questionnaires were performed, as described in Onderwater et al. 21 In short, during the 20-minute GTN infusion, headache characteristics and associated symptoms were documented every 5 minutes. After the infusion period, the occurrence of premonitory symptoms, headache, and associated symptoms was documented every 15 minutes until 5 hours after GTN infusion. After the study day (6 hours after GTN infusion), to determine GTN responder status, participants filled in a headache diary and were asked for headache fitting migraine-like attack onset in a telephone follow-up ~3 days after participation. Headache intensity was scored with a verbal rating scale (VRS) from 0 to 10 (0 indicating no headache, 1 indicating a very mild headache and 10 indicating the worst possible headache pain imaginable). In addition, the response form included the type of pain, localization, associated symptoms, premonitory symptoms, and adverse events. Furthermore, subjects with migraine were asked whether the reported headache resembled their usual migraine attacks. Despite the resemblance with spontaneous attacks, induced attacks are referred to as ‘migraine-like headaches’, as they cannot fulfil all criteria of a migraine without aura attack; for this the attack needs to be spontaneous and last (untreated) at least 4 hours.3 Therefore, in accordance with earlier provocation studies,16 migraine-like attack onset (ictal) was determined as either (1) a moderate to severe headache (VRS ≥ 4) fulfilling ICHD-3 criteria C and D for migraine without aura or (2) a headache described as mimicking the subject’s usual migraine attack and treated with acute migraine medication. PGE2 quantification PGE2 was quantified in EDTA plasma using a method analogue for the quantification of 8-isoPGF2α, previously described.35 In short, 250 mL EDTA plasma was diluted with 2.0 mL sodium acetate buffer (0.1 M, pH 3.5) and 3 mL PGE2-d4 (50 ng/mL) in methanol (MeOH) was added. The samples were loaded onto C18 SPE cartridges (200 mg, 3cc; Waters, Sep-Pak, Milford, MA) that had been conditioned and equilibrated with MeOH and water. After a wash with water and n-hexane samples were eluted using methyl formate. Eluates were then dried under a gentle stream of nitrogen at 40°C and reconstituted in 150 mL 40% MeOH. Samples were measured by Liquid Chromatography (Shimadzu SIL-30AC autosampler, two Shimadzu LC-30AD pumps and a Shimadzu CTO-20AC column oven) coupled to a Sciex Qtrap 6500 mass spectrometer. Forty-mL samples were injected and separated on a C18 column
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