2 CHAPTER 2 32 Methods Study population The study included participants from the Erasmus Rucphen Family (ERF) study 37, 38. This study population is based on a genetically isolated community in the Southwest of the Netherlands. In brief, the ERF study population includes 3,465 living descendants of 22 couples that had at least six children baptized in the community church between 1850 and 1900. Hence, study participants were all members of a large extended pedigree and all of European ancestry. All individuals 18 years and older were invited to participate. Migraine diagnoses Migraine was diagnosed using a validated three-stage screening procedure,2 based on International Classification of Headache Disorder formerly ICHD-II, now ICHD-III criteria.3, 39 Details on the migraine case-finding procedure have been published previously.36 In short, first, participants filled out a five-item screening questionnaire on headache and aura symptoms. Next, screenpositives completed an additional detailed questionnaire on headache and aura symptoms. Finally, the diagnosis was validated with a telephone interview by a physician trained in headache disorders. Probable migraine patients were excluded. ERF participants who were negative for severe headache and/or migraine based on the aforementioned three-stage screening procedure were included as controls.2, 36 Samples from participants were collected after overnight fasting. 1H-NMR spectroscopy metabolite profiling: data processing and quality control Venous blood samples had been drawn by venipuncture from the median cubital vein from participants of the ERF study after at least 8 h fasting period. Samples were centrifuged at 1,000-2,000 x g for 10 minutes at 4°C and serum was aliquoted in cryovials and stored at -80°C until further use. The 1H-NMR data were generated as part of a larger project and described by Vaarhorst et al.40 All 1H-NMR spectroscopy experiments had been acquired on a 600 MHz Bruker Avance II spectrometer (Bruker) equipped with a 5-mm triple resonance inverse (TCI) cryogenic probe head with Z-gradient system and automatic tuning and matching. All experiments were recorded at 310K.Temperature calibration was done prior to each batch of measurements using the method of Findeisen et al.41 The duration of the π/2 pulses were automatically calibrated for each individual sample using a homonuclear-gated nutation experiment on the locked and shimmed samples after automatic tuning and matching of the probe head.42 Then, stored samples were thawed at 4°C and mixed by inverting the containers ten times. Samples (300 µL) were mixed with 300 µL 75 mM disodium phosphate buffer in H2O/D2O (80/20) (pH 7.4), containing 6.15 mM NaN3 and 4.64 mM sodium 3-[trimethylsilyl] d4-propionate (TSP), using a Gilson 215 liquid handler in combination with a Bruker SampleTrack system (Bruker, Karlsruhe, Germany). Samples were transferred into 5-mm SampleJet NMR tubes (Bruker) in 96-tube racks using a modified Gilson 215 tube filling station (Gilson, Middleton, WI, USA) and kept at 6°C on a SampleJet sample changer (Bruker) while queued for acquisition.
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