title: Rapid, simultaneous quantitation of mono and dioxygenated metabolites of arachidonic acid in human CSF and rat brain.
abstract: Currently, there are few biomarkers to predict the risk of symptomatic cerebral vasospasm (SV) in subarachnoid hemorrhage (SAH) patients. Mono and dioxygenated arachidonic acid metabolites, involved in the pathogenesis of ischemic injury, may serve as indicators of SV. This study developed a quantitative UPLC-MS/MS method to simultaneously measure hydroxyeicosatetraenoic acid (HETE), dihydroxyeicosatrienoic acid (DiHETrE), and epoxyeicosatrienoic acid (EET) metabolites of arachidonic acid in cerebrospinal fluid (CSF) samples of SAH patients. Additionally, we determined the recovery of these metabolites from polyvinylchloride (PVC) bags used for CSF collection. Linear calibration curves ranging from 0.208 to 33.3 ng/ml were validated. The inter-day and intra-day variance was less than 15% at most concentrations with extraction efficiency greater than 73%. The matrix did not affect the reproducibility and reliability of the assay. In CSF samples, peak concentrations of 8,9-DiHETrE, 20-HETE, 15-HETE, and 12-HETE ranged from 0.293 to 24.9 ng/ml. In rat brain cortical tissue samples, concentrations of 20-, 15-, 12-HETE, 8,9-EET, and 14,15-, 11,12-DiHETrE ranged from 0.57 to 23.99 pmol/g wet tissue. In rat cortical microsomal incubates, all 10 metabolites were measured with formation rates ranging from 0.03 to 7.77 pmol/mg/min. Furthermore, 12-HETE and EET metabolites were significantly altered by contact with PVC bags at all time points evaluated. These data demonstrate that the simultaneous measurement of these compounds in human CSF and rat brain can be achieved with a UPLC-MS/MS system and that this method is necessary for evaluation of these metabolites as potential quantitative biomarkers in future clinical trials.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Tricia M
authors.initials: TM
authors.lastname: Miller
authors.forename: Mark K
authors.initials: MK
authors.lastname: Donnelly
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Crago
authors.forename: Dana M
authors.initials: DM
authors.lastname: Roman
authors.forename: Paula R
authors.initials: PR
authors.lastname: Sherwood
authors.forename: Michael B
authors.initials: MB
authors.lastname: Horowitz
authors.forename: Samuel M
authors.initials: SM
authors.lastname: Poloyac
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000134:cerebrospinal fluid / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000134:cerebrospinal fluid / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000134:cerebrospinal fluid / Q000378:metabolism; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D013345:Subarachnoid Hemorrhage / Q000134:cerebrospinal fluid / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D020301:Vasospasm, Intracranial / Q000134:cerebrospinal fluid
chemical_list: D006893:Hydroxyeicosatetraenoic Acids; D016718:Arachidonic Acid; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; C050715:8,9-epoxyeicosatrienoic acid; D015126:8,11,14-Eicosatrienoic Acid