20-HETE is associated with unfavorable outcomes in subarachnoid hemorrhage patients.

Emerging evidence has suggested that patients experiencing aneurysmal subarachnoid hemorrhage (aSAH) develop vascular dysregulation as a potential contributor to poor outcomes. Preclinical studies have implicated the novel microvascular constrictor, 20-hydroxyeicosatetraenoic acid (20-HETE) in aSAH pathogenesis, yet the translational relevance of 20-HETE in patients with aSAH is largely unknown. The goal of this research was to determine the relationship between 20-HETE cerebrospinal fluid (CSF) levels, gene variants in 20-HETE synthesis, and acute/long-term aSAH outcomes. In all, 363 adult patients (age 18 to 75) with aSAH were prospectively recruited from the University of Pittsburgh Medical Center neurovascular Intensive Care Unit. Patients were genotyped for polymorphic variants and cytochrome P450 (CYP)-eicosanoid CSF levels were measured over 14 days. Outcomes included delayed cerebral ischemia (DCI), clinical neurologic deterioration (CND), and modified Rankin Scores (MRS) at 3 and 12 months. Patients with CND and unfavorable 3-month MRS had 2.2- and 2.7-fold higher mean 20-HETE CSF levels, respectively. Patients in high/moderate 20-HETE trajectory groups (35.7%) were 2.5-, 2.1-, 3.1-, 3.3-, and 2.1-fold more likely to have unfavorable MRS at 3 months, unfavorable MRS at 12 months, mortality at 3 months, mortality at 12 months, and CND, respectively. These results showed that 20-HETE is associated with acute and long-term outcomes and suggest that 20-HETE may be a novel target in aSAH.

Protective role of p450 epoxyeicosanoids in subarachnoid hemorrhage.

BACKGROUND: Patients recovering from aneurysmal subarachnoid hemorrhage (SAH) are at risk for developing delayed cerebral ischemia (DCI). Experimental and human studies implicate the vasoconstrictor P450 eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE) in the pathogenesis of DCI. To date, no studies have evaluated the role of vasodilator epoxyeicosatrienoic acids (EETs) in DCI. METHODS: Using mass spectrometry, we measured P450 eicosanoids in cerebrospinal fluid (CSF) from 34 SAH patients from 1 to 14 days after admission. CSF eicosanoid levels were compared in patients who experienced DCI versus those who did not. We then studied the effect of EETs in a model of SAH using mice lacking the enzyme soluble epoxide hydrolase (sEH), which catabolizes EETs into their inactive diol. To assess changes in vessel morphology and cortical perfusion in the mouse brain, we used optical microangiography, a non-invasive coherence-based imaging technique. RESULTS: Along with increases in 20-HETE, we found that CSF levels of 14,15-EET were elevated in SAH patients compared to control CSF, and levels were significantly higher in patients who experienced DCI compared to those who did not. Mice lacking sEH had elevated 14,15-EET and were protected from the delayed decrease in microvascular cortical perfusion after SAH, compared to wild type mice. CONCLUSIONS: Our findings suggest that P450 eicosanoids play an important role in the pathogenesis of DCI. While 20-HETE may contribute to the development of DCI, 14,15-EET may afford protection against DCI. Strategies to enhance 14,15-EET, including sEH inhibition, should be considered as part of a comprehensive approach to prevent DCI.

Genetic markers in the EET metabolic pathway are associated with outcomes in patients with aneurysmal subarachnoid hemorrhage.

Preclinical studies show that epoxyeicosatrienoic acids (EETs) regulate cerebrovascular tone and protect against cerebral ischemia. We investigated the relationship between polymorphic genes involved in EET biosynthesis/metabolism, cytochrome P450 (CYP) eicosanoid levels, and outcomes in 363 patients with aneurysmal subarachnoid hemorrhage (aSAH). Epoxyeicosatrienoic acids and dihydroxyeicosatetraenoic acid (DHET) cerebrospinal fluid (CSF) levels, as well as acute outcomes defined by delayed cerebral ischemia (DCI) or clinical neurologic deterioration (CND), were assessed over 14 days. Long-term outcomes were defined by Modified Rankin Scale (MRS) at 3 and 12 months. CYP2C8*4 allele carriers had 44% and 36% lower mean EET and DHET CSF levels (P=0.003 and P=0.007) and were 2.2- and 2.5-fold more likely to develop DCI and CND (P=0.039 and P=0.041), respectively. EPHX2 55Arg, CYP2J2*7, CYP2C8*1B, and CYP2C8 g.36785A allele carriers had lower EET and DHET CSF levels. CYP2C8 g.25369T and CYP2C8 g.36755A allele carriers had higher EET levels. Patients with CYP2C8*2C and EPHX2 404del variants had worse long-term outcomes while those with EPHX2 287Gln, CYP2J2*7, and CYP2C9 g.816G variants had favorable outcomes. Epoxyeicosatrienoic acid levels were associated with Fisher grade and unfavorable 3-month outcomes. Dihydroxyeicosatetraenoic acids were not associated with outcomes. No associations passed Bonferroni multiple testing correction. These are the first clinical data demonstrating the association between the EET biosynthesis/metabolic pathway and the pathophysiology of aSAH.

Plasma platelet-activating factor-acetyl hydrolase activity and the levels of free forms of biomarker of lipid peroxidation in cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage.

BACKGROUND: Free radicals and lipid peroxidation are thought to be related to the vasospasm generation after subarachnoid hemorrhage (SAH). Plasma platelet-activating factor-acetyl hydrolase (PAF-AH) degrades phospholipids with an oxidatively modified fatty acyl chain. OBJECTIVE: To compare plasma PAF-AH activity and free forms of biomarker of lipid peroxidation in cerebrospinal fluid (CSF) between patients with and without symptomatic vasospasm (SVS) after SAH. METHODS: The identification of PAF-AH in CSF was performed by Western blotting. The genotype at position 279 of the plasma PAF-AH gene was determined. The activities of PAF-AH and the levels of free 8-iso-prostaglandin F2α (free isoPs), free hydroxyoctadecadienoic acid (free HODE), and free hydroxyeicosatetraenoic acid (free HETE) in CSF were measured. RESULTS: The PAF-AH in CSF was confirmed to be only the plasma type. The genotype of the plasma PAF-AH was not different between patients with and without SVS. Free isoPs, free HODE, and free HETE showed higher values in patients without SVS in 0 to 4 days and 5 to 9 days after SAH. The PAF-AH activity also was higher in patients without SVS in 0 to 4 days and 5 to 9 days after SAH. The associations between PAF-AH activity and free isoPs, and between PAF-AH activity and free HODE were significant. CONCLUSION: Oxidized lipids of lipoproteins and blood cell membranes produced by reactive oxygen species in CSF when SAH occurs may be the main source of lipid peroxidation. Plasma PAF-AH can hydrolyze oxidized phospholipids, and may attenuate the spreading of lipid peroxidation and participate in defense mechanisms against vasospasm after SAH.

Cerebrospinal fluid 20-HETE is associated with delayed cerebral ischemia and poor outcomes after aneurysmal subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Delayed cerebral ischemia (DCI) is a major complication after aneurysmal subarachnoid hemorrhage (aSAH); it is manifested by changes in cerebral blood flow accompanied by neurological decline, and it results in long-term functional and neuropsychological impairment. Preclinical evidence has demonstrated that the arachidonic acid metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), affects cerebral microvascular tone and cerebral blood flow after aSAH. The purpose of this study was to determine whether cerebrospinal fluid 20-HETE levels were associated with DCI and long-term neuropsychological outcomes in aSAH patients. METHODS: Cerebrospinal fluid samples were collected twice daily through 14 days after hemorrhage on 108 acute, adult, aSAH patients. Samples were analyzed for 20-HETE via HPLC MSQ single quadrupole mass spectrometry. DCI was defined as the presence of impaired cerebral blood flow (angiographic vasospasm, elevated transcranial Dopplers, abnormal computed tomography or magnetic resonance perfusion scans) accompanied by neurological deterioration. Outcomes, including death and neuropsychological testing, were completed at 3 months after hemorrhage. RESULTS: Detectable 20-HETE levels were observed in 31% of patient samples and were associated with severity of hemorrhage (Hunt & Hess [HH], P=0.04; Fisher, P=0.05). Detection of 20-HETE was not associated with angiographic vasospasm (P=0.34); however, detectable 20-HETE was significantly associated with DCI (P=0.016). Our data also suggest that detectable 20-HETE was associated with decreased performance in 5 neuropsychological domains. CONCLUSIONS: These results provide the first clinical evidence that cerebrospinal fluid 20-HETE concentrations are associated with DCI and poor outcomes, and this provides impetus for future studies to elucidate the clinical utility of inhibiting 20-HETE formation as a novel therapeutic intervention in patients with aSAH.

Lipid mediators in cerebral spinal fluid of traumatic brain injured patients.

BACKGROUND: Enzymatic and nonenzymatic oxidation of polyunsaturated fatty acids leads to the formation of biologically active products known as lipid mediators. In the brain, lipid mediators play an important role in supporting homeostasis and normal function. Thus, levels of these metabolites in normal and pathologic conditions in the brain are particularly relevant in understanding the transition to disease. METHODS: In this study, liquid chromatography tandem mass spectrometry was used to analyze lipid mediators in cerebrospinal fluid (CSF) of controls and traumatic brain injured (TBI) patients. RESULTS: Our results showed that the levels of arachidonic acid (AA), docosahexaenoic acid (DHA), 5- and 12- eicosatetraenoic acid (HETE) were significantly increased in the CSF of TBI patients. The magnitude of increase was 10-fold for AA, DHA, and 5-HETE and 17-fold for 12-HETE. Prostaglandins and leukotrienes were not detected in CSF of either control or brain injured patients. Furthermore, this study found that isoprostanes and thromboxanes are present in CSF of brain injured patients. CONCLUSIONS: This study clearly shows that certain lipid mediators accumulate in the CSF of TBI patient. This study also suggests the potential use of DHA, AA, 5- and 12-HETE as biochemical markers of brain injury and to monitor the impact of interventions.

Rapid, simultaneous quantitation of mono and dioxygenated metabolites of arachidonic acid in human CSF and rat brain.

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.

Elevated cerebrospinal fluid levels of prostaglandin E2 and 15-(S)-hydroxyeicosatetraenoic acid in multiple sclerosis.

OBJECTIVE: To test the hypothesis that the arachodinic acid metabolites prostaglandin E2 (PGE2) and 15-(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in cerebrospinal fluid (CSF) are elevated and reflect neuroinflammation and degenerative changes in multiple sclerosis (MS). PATIENTS AND METHODS: We measured PGE2 and 15(S)-HETE concentrations, as well as markers of axonal and astroglial injury in CSF from 46 MS patients, 46 healthy siblings and 50 controls. RESULTS: We found elevated levels of both PGE2 and 15(S)-HETE in MS compared with the control and sibling groups. Siblings had lower PGE2 levels and higher 15(S)-HETE levels than controls. There were no correlations between either PGE2 or 15(S)-HETE and clinical scores of MS severity or biochemical markers of axonal or astroglial injury. CONCLUSION: These data suggest no direct involvement of PGE2 and 15(S)-HETE in the MS disease process. Rather, the elevated levels reflect a general up-regulation of arachidonic acid metabolism and neuroinflammation.

Reversal of delayed vasospasm by TS-011 in the dual hemorrhage dog model of subarachnoid hemorrhage.

PURPOSE: Arachidonic acid is avidly metabolized to a potent vasoconstrictor, 20-hydroxyeicosatetraenoic acid (20-HETE), in the cerebral circulation. 20-HETE has been reported to contribute to the acute fall in cerebral blood flow following subarachnoid hemorrhage (SAH), but its role in the development of delayed vasospasm is unknown. The present study examined whether delayed vasospasm is associated with elevations in 20-HETE in CSF in the dual hemorrhage model of SAH in dogs and if blockade of the synthesis of 20-HETE with N-(3-chloro-4-morpholin-4-yl)phenyl-N'-hydroxyimido formamide (TS-011) can reverse delayed vasospasm in this model. MATERIALS AND METHODS: Delayed vasospasm was induced in 22 adult beagle dogs by dual injection of blood (0.5 mL/kg) into the cisterna magna on days 1 and 4. Sequential samples of CSF were collected before intracisternal injections of blood on days 1 and 4 and after the development of delayed vasospasm on day 7. Sequential angiograms were obtained before and after intracisternal injection of blood on days 1 and 4 and before and 1 hour after administration of TS-011 (1 mg/kg IV) on day 7. RESULTS: The dogs consistently developed delayed vasospasm, and the diameter of the basilar artery fell to 68 +/- 3% (n = 15), 3 days after the second intracisternal injection of blood. The levels of 20-HETE in CSF increased from 4 +/- 2 to 39 +/- 16 pg/mL. In 9 dogs with delayed vasospasm, acute blockade of the synthesis of 20-HETE with TS011 (1 mg/kg IV) significantly increased the diameter of the basilar artery by 39%. Chronic administration of TS-011 (1 mg/kg per day) attenuated the development of delayed vasospasm, and the diameter of the basilar artery fell by 17 +/- 1% versus the 33 +/- 3% decrease in diameter seen in control animals 3 days following the second injection of blood into the cisterna magna. CONCLUSIONS: These results indicate that the development of delayed vasospasm in dogs is associated with an increase in 20-HETE levels in CSF, and acute blockade of the synthesis of 20-HETE with TS-011 reverses delayed vasospasm in this model.

Elevation of 12/15 lipoxygenase products in AD and mild cognitive impairment.

The 12/15 lipoxygenase (12/15LOX) enzyme is increased in pathologically affected frontal and temporal regions of Alzheimer's disease (AD) brains compared with controls. Herein, we measured 12(S)-HETE and 15(S)-HETE levels, products of 12/15LOX, in cerebrospinal fluid (CSF) of normal individuals, subjects with mild cognitive impairment (MCI) and AD. Compared with controls, there was a significant increase of both metabolites in CSF from AD and MCI, which correlated with lipid peroxidation and tau protein levels. These results suggest that the activation of this enzyme occurs early in the course of AD, before the onset of overt dementia, thereby implicating 12/15LOX-mediated lipid peroxidation in the pathogenesis of AD.

Identification and quantification of the hydroxyeicosatetraenoic acids, 20-HETE and 12-HETE, in the cerebrospinal fluid after subarachnoid hemorrhage.

PURPOSE: The monohydroxylated metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), is a potent vasoconstrictor of cerebral microvessels. 20-HETE formation is substantially elevated in the cerebral spinal fluid (CSF) in the rat subarachnoid hemorrhage (SAH) model. The presence of 20-HETE in human CSF has not been demonstrated. Therefore, it was the purpose of this study to determine if HETE metabolites are present in human CSF after SAH. METHODS: CSF samples were collected daily from four SAH patients over 15 days. HETE metabolites were separated by HPLC with identification by ion-trap MS/MS and quantification via single quadrupole MS operating in negative single ion monitoring mode. RESULTS: Two major metabolites were identified as 12-HETE and 20-HETE. 20-HETE maximal concentrations were 2.9 and 0.7 ng/ml at approximately 70 h in the two patients with symptomatic cerebral vasospasm (SV) after SAH. Concentrations of 12-HETE in these patients peaked at 21.9 ng/ml and 2.8 ng/ml. Concentrations of 20-HETE and 12-HETE were non-detectible in the majority of the samples obtained from two matched SAH patients without SV. CONCLUSIONS: This study is the first to demonstrate that 20-HETE and 12-HETE are present in the CSF of SAH patients at physiologically relevant concentrations. Based on this information future prospective studies will allow for the delineation of the role of these metabolites in the pathogenesis of SAH.

Contribution of 5-hydroxytryptamine1B receptors and 20-hydroxyeiscosatetraenoic acid to fall in cerebral blood flow after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: This study examined the interaction between 5-hydroxytryptamine1B (5-HT1B) receptors and 20-hydroxyeiscosatetraenoic acid (20-HETE) in contributing to the acute fall in regional cerebral blood flow (rCBF) after subarachnoid hemorrhage (SAH) in rats. METHODS: The effects of intracisternal injection of 0.3 mL of arterial blood, artificial cerebrospinal fluid, and 5-HT on rCBF and the levels of 20-HETE and 5-HT in cerebrospinal fluid were measured in rats pretreated with vehicle, a 5-HT1B receptor antagonist (isamoltane hemifumarate), or an inhibitor of the synthesis of 20-HETE (HET0016). The effects of HET0016 and isamoltane on the vasoconstrictor response and changes in [Ca2+]i to 5-HT were also studied in middle cerebral arteries and vascular smooth muscle cells isolated from these vessels. RESULTS: 20-HETE and 5-HT levels in cerebrospinal fluid rose from 172+/-10 to 629+/-44 ng/mL and from 6+/-4 to 1163+/-200 nmol/mL, respectively, after SAH. rCBF fell by 30% 10 minutes after SAH, and it remained at this level for the next 2 hours. Blockade of 5-HT1B receptors prevented the sustained fall in rCBF seen after SAH. Intracisternal injection of 5-HT mimicked SAH by increasing 20-HETE levels in cerebrospinal fluid to 475+/-94 ng/mL and reducing rCBF by 30%. Blockade of the synthesis of 20-HETE with HET0016 prevented the fall in rCBF produced by 5-HT. Isamoltane and HET0016 reduced the vasoconstrictor response of isolated MCA to 5-HT by >60% and diminished the rise in [Ca2+]i produced by 5-HT in vascular smooth muscle cells isolated from these arteries. CONCLUSIONS: These results suggest that the release of 5-HT after SAH activates 5-HT1B receptors and the synthesis of 20-HETE and that 20-HETE contributes to the acute fall in rCBF by potentiating the vasoconstrictor response of cerebral vessels to 5-HT.

Participation of lipoxygenase products from arachidonic acid in the pathogenesis of cerebral vasospasm.

To examine the possible involvement of lipoxygenase products from arachidonic acid in the pathogenesis of delayed vasospasm after subarachnoid hemorrhage (SAH), we measured the contents of hydroxyeicosatetraenoic acids (HETEs) in the subarachnoid clot, the cerebrospinal fluid, and the basilar artery, using the canine "two-hemorrhage" model. Lipoxygenase activity in the subarachnoid clot and the basilar artery was measured, ex vivo, using samples obtained 7 days after SAH. For a quantitative analysis of HETEs, each sample was homogenized with either ice-cold saline or methanol. The lipid extract was then submitted to reverse-phase HPLC. The identity of each HETE was further confirmed using straight-phase HPLC and gas chromatography-mass spectrometry. When the basilar artery was homogenized with ice-cold saline, a significant increase in the 5-HETE content was observed on SAH day 8. However, when the artery was homogenized with methanol, HETEs were not detected. In the case of incubation in the presence of arachidonic acid and calcium ionophore A23187, the 5-lipoxygenase activity was remarkably increased in the basilar artery exposed to SAH, compared to that of normal dogs. The subarachnoid clot contained a significant amount of 12-HETE (average 1.8 nmol/g wet weight) from day 2 to day 8. The administration of 1,2-bis(nicotinamido)propane significantly ameliorated vasospasm in the two-hemorrhage model, simultaneously inhibiting the 5-lipoxygenase activity of the basilar artery. Our observations show that the activities of 12- and 5-lipoxygenases are significantly increased after SAH in the subarachnoid clot and the basilar artery, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Eicosanoids in human ventricular cerebrospinal fluid following severe brain injury.

Recent evidence has shown that a variety of prostaglandins and leukotrienes can be produced in brain tissue after injury in animals. It has also been speculated that increases in brain prostaglandins occur in humans following injury. Ventricular cerebrospinal fluid (CSF) samples have been obtained from children with static lesions (controls) as well as children with acute brain injury and eicosanoids measured by immunologic techniques. Metabolites of prostacyclin (6-keto-PGF1 a) and thromboxane A2 (thromboxane B2) were the major eicosanoids found in CSF, and levels of these compounds were increased 3-10 times in acutely injured patients. Prostaglandin E2 was also found in lower amounts, although in one case its level was very high. Prostaglandin D2 was also present, but in low amounts. No leukotrienes were found in CSF samples that were purified by HPLC prior to immunoassay. Elevated levels of hydroxyeicosatetraenoic acids (HETEs) were observed in those samples stored frozen, but these metabolites were most probably due to autooxidation of arachidonic acid in CSF. Arachidonic acid concentration in CSF was typically found to be in the range of 10-200 ng/ml, but was found to be 5-10 fold higher in one severely injured patient. Thus, elevated free arachidonic acid and various oxygenated metabolites were observed in CSF following brain injury.

Thromboxane and 5-HETE increase after experimental spinal cord injury in rabbits.

Eicosanoids are known mediators of inflammation, vascular permeability, and are involved in microcirculatory blood flow regulation. To study their potential involvement in the pathophysiology of CNS trauma we used a rabbit spinal cord trauma model. Rabbits were subjected to lumbar spinal cord trauma produced by a modification of the Allen weight-drop method. TXB2, 6-keto-PGF1 alpha, PGE2, and 5-hydroxyeicosatetraenoic acid (5-HETE) release from spinal cord slices incubated ex vivo were measured by radioimmunoassay at 5, 30 min, 24 hrs, and 2 wks after trauma. Five and 30 min after trauma the TXB2/6-keto-PGF1 alpha ratio was elevated and the release of 5-HETE at 5 min after trauma increased in the injured spinal cord whereas release of 6-keto-PGF1 alpha and PGE2 remained at base-line levels. In the thoracic spinal cord, TXB2 and 6-keto-PGF1 alpha release were increased at 30 min after trauma. Release of 5-HETE from the injured spinal cord was also elevated 24 hrs after trauma. Two wks after trauma, TXB2 and 6-keto-PGF1 alpha release were also elevated in the injured spinal cord. Measurements of tissue water content by microgravimetry indicated progressive edema in the injury site while histopathological evaluation indicated progressive damage and tissue destruction. The results of this study suggest that eicosanoids may be involved in the pathophysiology of spinal cord trauma through two potential mechanisms: 1) site specific increase in the TXB2/6-keto-PGF1 alpha ratio immediately following trauma which is due primarily to an increase in TXA2 synthesis; 2) the increase synthesis of 5-HETE which signals the activation of the 5-lipoxygenase pathway of arachidonate metabolism and production of mediators that are involved in inflammatory mechanisms and may affect local blood flow regulation and blood-spinal cord barrier integrity.