3 CHAPTER 3 52 Methods Study Design One aim of this study is to determine to what extent amine levels in blood plasma correlate to those in CSF from healthy controls. To this end, we examined: (1) single-metabolite correlations of amine levels between CSF and plasma, and (2) plasma/CSF ratio-metabolite correlations. In addition, we examined both types of correlation in a paroxysmal CNS disease, namely migraine, as to investigate if correlations can provide insight into disease pathophysiology. The study was conducted according to the criteria of the Declaration of Helsinki and was approved by the Leiden University Medical Center institutional ethics committee. All participants provided written informed consent prior to the study. Healthy controls Participants were recruited as healthy controls in a larger biochemical study on migraine.13 CSF and plasma were collected purely for research purposes. Exclusion criteria for healthy controls were severe neurological (including migraine or other high-frequent headache disorder) or psychiatric disorders, any oncological history, or contraindication for lumbar puncture (including signs and symptoms of increased intracranial pressure, local skin infection, or coagulopathy, including the use of anti-coagulant drugs or platelet inhibitors). Migraine participants Participants with episodic migraine were recruited as part of the same study as that of the healthy controls. Migraine was diagnosed according to the International Classification of Headache Disorders.14 CSF and plasma were collected outside migraine attacks (interictal) 4, 5, 13. Apart from migraine diagnosis the same exclusion criteria were used for the healthy controls. Sample collection CSF sampling was performed via lumbar puncture before 13.00 p.m. and after overnight fasting (participants were only allowed to drink water during 8 h prior to sampling). Lumbar puncture was performed between the L3/L4, L4/L5 or L5/S1 interspace using a .9 (20 G) x 90 mm Quincke traumatic needle (MediPlast, Malmö, Sweden) or .9 (20 G), .7 (22 G), or .5 (24 G) x 90 mm Sprotte atraumatic needle (Pajunk, Geisingen, Germany). Intracranial pressure was measured, and first 3 mL CSF was sampled for routine diagnostics (cell count, glucose and total protein levels), prior CSF collection for metabolic measurements. For metabolomics measurements 3.8 mL of CSF was sampled directly in a 15-mL polypropylene falcon tube (tube P1a; Cat. No. 188 271, (Greiner Bio-One, Alphen aan de Rijn, The Netherlands) and centrifuged at 4°C for 5 min (2000 rpm, 747 g).15 The supernatant was transferred to a new 15-mL polypropylene falcon tube, inverted several times, and divided in .5 mL aliquots into 1.8-mL NuncTM cryotubes (Cat. No.
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