A rapid UPLC-MS/MS assay for eicosanoids in human plasma: Application to evaluate niacin responsivity.

A rapid and sensitive method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to simultaneously quantify hydroxyeicosatetraenoic (HETE), dihydroxyeicosatrienoic (DiHETrE), epoxyeicosatrienoic acid (EET), and prostaglandin metabolites of arachidonic acid in human plasma. Sample preparation consisted of solid phase extraction with Oasis HLB (30mg) cartridges for all metabolites. Separation of HETEs, EETs, and DiHETrEs was achieved on an Acquity UPLC BEH C18, 1.7µm (100×2.1mm) reversed-phase column (Waters Corp, Millford, MA) with negative electrospray ionization mass spectrometric detection. A second injection of the same extracted sample allowed for separation and assessment of prostaglandin metabolites under optimized UPLC-MS/MS conditions. Additionally, the endogenous levels of these metabolites in five different matrices were determined in order to select the optimal matrix for assay development. Human serum albumin was shown to have the least amount of endogenous metabolites, a recovery efficiency of 79-100% and a matrix effect of 71 - 100%. Linear calibration curves ranging from 0.416 to 66.67ng/ml were validated. Inter-assay and intra-assay variance was less than 15% at most concentrations. This method was successfully applied to quantify metabolite levels in plasma samples of healthy control subjects receiving niacin administration to evaluate the association between niacin administration and eicosanoid plasma level response.

Hepatic Overexpression of CD36 Improves Glycogen Homeostasis and Attenuates High-Fat Diet-Induced Hepatic Steatosis and Insulin Resistance.

The common complications in obesity and type 2 diabetes include hepatic steatosis and disruption of glucose-glycogen homeostasis, leading to hyperglycemia. Fatty acid translocase (FAT/CD36), whose expression is inducible in obesity, is known for its function in fatty acid uptake. Previous work by us and others suggested that CD36 plays an important role in hepatic lipid homeostasis, but the results have been conflicting and the mechanisms were not well understood. In this study, by using CD36-overexpressing transgenic (CD36Tg) mice, we uncovered a surprising function of CD36 in regulating glycogen homeostasis. Overexpression of CD36 promoted glycogen synthesis, and as a result, CD36Tg mice were protected from fasting hypoglycemia. When challenged with a high-fat diet (HFD), CD36Tg mice showed unexpected attenuation of hepatic steatosis, increased very low-density lipoprotein (VLDL) secretion, and improved glucose tolerance and insulin sensitivity. The HFD-fed CD36Tg mice also showed decreased levels of proinflammatory hepatic prostaglandins and 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictive and proinflammatory arachidonic acid metabolite. We propose that CD36 functions as a protective metabolic sensor in the liver under lipid overload and metabolic stress. CD36 may be explored as a valuable therapeutic target for the management of metabolic syndrome.

The cytochrome P450 epoxygenase pathway regulates the hepatic inflammatory response in fatty liver disease.

Fatty liver disease is an emerging public health problem without effective therapies, and chronic hepatic inflammation is a key pathologic mediator in its progression. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory effects. Although promoting the effects of EETs elicits anti-inflammatory and protective effects in the cardiovascular system, the contribution of CYP-derived EETs to the regulation of fatty liver disease-associated inflammation and injury is unknown. Using the atherogenic diet model of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), our studies demonstrated that induction of fatty liver disease significantly and preferentially suppresses hepatic CYP epoxygenase expression and activity, and both hepatic and circulating levels of EETs in mice. Furthermore, mice with targeted disruption of Ephx2 (the gene encoding soluble epoxide hydrolase) exhibited restored hepatic and circulating EET levels and a significantly attenuated induction of hepatic inflammation and injury. Collectively, these data suggest that suppression of hepatic CYP-mediated EET biosynthesis is an important pathological consequence of fatty liver disease-associated inflammation, and that the CYP epoxygenase pathway is a central regulator of the hepatic inflammatory response in NAFLD/NASH. Future studies investigating the utility of therapeutic strategies that promote the effects of CYP-derived EETs in NAFLD/NASH are warranted.

Rapid and simultaneous quantitation of prostanoids by UPLC-MS/MS in rat brain.

The metabolites of arachidonic acid (AA) produced from the cyclooxygenase (COX) pathway, collectively termed as prostanoids, and from the CYP 450 pathway, eicosanoids, have been implicated in various neuro-degenerative and neuroinflammatory diseases. This study developed a quantitative UPLC-MS/MS method to simultaneously measure 11 prostanoids including prostaglandins and cyclopentenone metabolites in the rat brain cortical tissue. Linear calibration curves ranging from 0.104 to 33.3ng/ml were validated. The inter-day and intra-day variance for all metabolites was less than 15%. The extraction recovery efficiency and matrix (deionized water) effects measured at 12.5ng/ml (750pg on column) ranged from 88 to 100% and 3 to 14%, respectively, with CV% values below 20%. Additionally, applying the processing and extraction conditions of this method to our previous CYP450 eicosanoids method resulted in overall improvement in extraction recovery and reduction in matrix effects at low (0.417ng/ml) and high (8.33ng/ml) concentrations. In rat brain cortical tissue samples, concentrations of prostanoids ranged from 10.2 to 937pmol/g wet tissue and concentration of eicosanoids ranged from 2.23 to 793pmol/g wet tissue. These data demonstrate that the successive measurement of prostanoids and eicosanoids from a single extracted sample of rat brain tissue can be achieved with a UPLC-MS/MS system and that this method is necessary for evaluation of these metabolites to delineate their role in various neuroinflammatory and cerebrovascular disorders.

Prostaglandin D2 toxicity in primary neurons is mediated through its bioactive cyclopentenone metabolites.

Prostaglandin D2 (PGD2) is the most abundant prostaglandin in brain but its effect on neuronal cell death is complex and not completely understood. PGD2 may modulate neuronal cell death via activation of DP receptors or its metabolism to the cyclopentenone prostaglandins (CyPGs) PGJ2, Δ(12)-PGJ2 and 15-deoxy-Δ(12,14)-PGJ2, inducing cell death independently of prostaglandin receptors. This study aims to elucidate the effect of PGD2 on neuronal cell death and its underlying mechanisms. PGD2 dose-dependently induced cell death in rat primary neuron-enriched cultures in concentrations of ≥10µM, and this effect was not reversed by treatment with either DP1 or DP2 receptor antagonists. Antioxidants N-acetylcysteine (NAC) and glutathione which contain sulfhydryl groups that can bind to CyPGs, but not ascorbate or tocopherol, attenuated PGD2-induced cell death. Conversion of PGD2 to CyPGs was detected in neuronal culture medium; treatment with these CyPG metabolites alone exhibited effects similar to those of PGD2, including apoptotic neuronal cell death and accumulation of ubiquitinated proteins. Disruption of lipocalin-type prostaglandin D synthase (L-PGDS) protected neurons against hypoxia. These results support the hypothesis that PGD2 elicits its cytotoxic effects through its bioactive CyPG metabolites rather than DP receptor activation in primary neuronal culture.

COX2-derived primary and cyclopentenone prostaglandins are increased after asphyxial cardiac arrest.

BACKGROUND: Cyclopentenone prostaglandins have been identified as potential neurotoxic agents in the setting of hypoxia-ischemia. Cyclooxygenase-2 (COX-2), the upstream enzyme responsible for prostaglandin production is upregulated following hypoxic-ischemic brain injury. However, the temporal production and concentration of cyclopentenone prostaglandins has not been described following global brain ischemia. METHODS: Global brain ischemia was induced in rats by asphyxial cardiac arrest (ACA) followed by resuscitation. Rats were sacrificed between 24h and 7 days following resuscitation and their brains removed. Western blot, immunohistochemistry, and mass spectroscopy were performed. A cohort of rats was pretreated with the COX-2 inhibitor SC58125. RESULTS: COX-2 is induced in hippocampus at 24h following ACA. Multiple prostaglandins, including cyclopentenone prostaglandin species, are increased in hippocampus as 24h following ACA. Prostaglandin and cyclopentenone prostaglandin concentrations are returned to baseline at 3 and 7 days post-ischemia. The COX-2 inhibitor SC58125 completely abrogates the post-ischemic increase in prostaglandins and cyclopentenone prostaglandins. CONCLUSIONS: Prostaglandins, including cyclopentenone prostaglandins, are increased in ischemic brain, peak at 24h and can be attenuated by the COX-2 inhibitor SC58125. These data establish the presence of potentially neurotoxic cyclopentenone prostaglandins in post-ischemic brains, thus identifying a target and therapeutic window for neuroprotective therapies.

Increased generation of cyclopentenone prostaglandins after brain ischemia and their role in aggregation of ubiquitinated proteins in neurons.

The cyclopentenone prostaglandin (CyPG) J2 series, including prostaglandin J2 (PGJ2), Δ¹²-PGJ2, and 15-deoxy-∆¹²,¹4-prostaglandin J2 (15d-PGJ2), are active metabolites of PGD2, exerting multiple effects on neuronal function. However, the physiologic relevance of these effects remains uncertain as brain concentrations of CyPGs have not been precisely determined. In this study, we found that free PGD2 and the J2 series CyPGs (PGJ2, Δ¹²-PGJ2, and 15d-PGJ2) were increased in post-ischemic rat brain as detected by UPLC-MS/MS with 15d-PGJ2 being the most abundant CyPG. These increases were attenuated by pre-treating with the cyclooxygenase (COX) inhibitor piroxicam. Next, effects of chronic exposure to 15d-PGJ2 were examined by treating primary neurons with 15d-PGJ2, CAY10410 (a 15d-PGJ2 analog lacking the cyclopentenone ring structure), or vehicle for 24 to 96 h. Because we found that the concentration of free 15d-PGJ2 decreased rapidly in cell culture medium, freshly prepared medium containing 15d-PGJ2, CAY10410, or vehicle was changed twice daily to maintain steady extracellular concentrations. Incubation with 2.5 µM 15d-PGJ2, but not CAY10410, increased the neuronal cell death without the induction of caspase-3 or PARP cleavage, consistent with a primarily necrotic mechanism for 15d-PGJ2-induced cell death which was further supported by TUNEL assay results. Ubiquitinated protein accumulation and aggregation was observed after 96 h 15d-PGJ2 incubation, accompanied by compromised 20S proteasome activity. Unlike another proteasome inhibitor, MG132, 15d-PGJ2 treatment did not activate autophagy or induce aggresome formation. Therefore, the cumulative cytotoxic effects of increased generation of CyPGs after stroke may contribute to delayed post-ischemic neuronal injury.

Enalapril reverses high-fat diet-induced alterations in cytochrome P450-mediated eicosanoid metabolism.

Metabolism of arachidonic acid by cytochrome P450 (CYP) to biologically active eicosanoids has been recognized increasingly as an integral mediator in the pathogenesis of cardiovascular and metabolic disease. CYP epoxygenase-derived epoxyeicosatrienoic and dihydroxyeicosatrienoic acids (EET + DHET) and CYP ω-hydroxylase-derived 20-hydroxyeicosatetraenoic acid (20-HETE) exhibit divergent effects in the regulation of vascular tone and inflammation; thus, alterations in the functional balance between these parallel pathways in liver and kidney may contribute to the pathogenesis and progression of metabolic syndrome. However, the impact of metabolic dysfunction on CYP-mediated formation of endogenous eicosanoids has not been well characterized. Therefore, we evaluated CYP epoxygenase (EET + DHET) and ω-hydroxylase (20-HETE) metabolic activity in liver and kidney in apoE(-/-) and wild-type mice fed a high-fat diet, which promoted weight gain and increased plasma insulin levels significantly. Hepatic CYP epoxygenase metabolic activity was significantly suppressed, whereas renal CYP ω-hydroxylase metabolic activity was induced significantly in high-fat diet-fed mice regardless of genotype, resulting in a significantly higher 20-HETE/EET + DHET formation rate ratio in both tissues. Treatment with enalapril, but not metformin or losartan, reversed the suppression of hepatic CYP epoxygenase metabolic activity and induction of renal CYP ω-hydroxylase metabolic activity, thereby restoring the functional balance between the pathways. Collectively, these findings suggest that the kinin-kallikrein system and angiotensin II type 2 receptor are key regulators of hepatic and renal CYP-mediated eicosanoid metabolism in the presence of metabolic syndrome. Future studies delineating the underlying mechanisms and evaluating the therapeutic potential of modulating CYP-derived EETs and 20-HETE in metabolic diseases are warranted.

Mild hypothermia decreases fentanyl and midazolam steady-state clearance in a rat model of cardiac arrest.

OBJECTIVES: Therapeutic hypothermia is widely employed for neuroprotection after cardiac arrest. However, concern regarding elevated drug concentrations during hypothermia and increased adverse drug reaction risk complicates concurrent pharmacotherapy. Many commonly used medications in critically ill patients rely on the cytochrome P450 3A isoform for their elimination. Therefore, our study objectives were to determine the effect of mild hypothermia on the in vivo pharmacokinetics of fentanyl and midazolam, two clinically relevant cytochrome P450 3A substrates, after cardiac arrest and to investigate the mechanisms of these alterations. DESIGN: Prospective, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Thirty-two adult male Sprague-Dawley rats. INTERVENTIONS: An asphyxial cardiac arrest rat model was used and mild hypothermia (33°C) was induced 1 hr post injury by surface cooling and continued for 10 hrs to mimic the prolonged clinical application of hypothermia accompanied by intensive care interventions. Fentanyl and midazolam were independently administered by intravenous infusion and plasma and brain concentrations were analyzed using ultraperformance liquid chromatography tandem mass spectrometry. Cytochrome P450 3a2 protein expression was measured and a Michaelis-Menten enzyme kinetic analysis was performed at 37°C and 33°C using control rat microsomes. MEASUREMENTS AND MAIN RESULTS: Mild hypothermia decreased the systemic clearance of both fentanyl (61.5 ± 11.5 to 48.9 ± 8.95 mL/min/kg; p < .05) and midazolam (89.2 ± 12.5 to 73.6 ± 12.1 mL/min/kg; p < .05) after cardiac arrest. The elevated systemic concentrations did not lead to parallel increased brain exposures of either drug. Mechanistically, no differences in cytochrome P450 3a2 expression was observed, but the in vitro metabolism of both drugs was decreased at 33°C vs. 37°C through reductions in enzyme metabolic capacity rather than substrate affinity. CONCLUSIONS: Mild hypothermia reduces the systemic clearances of fentanyl and midazolam in rats after cardiac arrest through alterations in cytochrome P450 3a2 metabolic capacity rather than enzyme affinity as observed with other cytochrome P450s. Contrasting effects on blood and brain levels further complicates drug dosing. Consideration of the impact of hypothermia on medications whose clearance is dependent on P450 3A metabolism is warranted.

Modification of ubiquitin-C-terminal hydrolase-L1 by cyclopentenone prostaglandins exacerbates hypoxic injury.

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ(12,14) -prostaglandin J(2) (15d-PGJ(2)), are active prostaglandin metabolites exerting a variety of biological effects that may be important in the pathogenesis of neurological diseases. Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain specific deubiquitinating enzyme whose aberrant function has been linked to neurodegenerative disorders. We report that [15d-PGJ(2)] detected by quadrapole mass spectrometry (MS) increases in rat brain after temporary focal ischemia, and that treatment with 15d-PGJ(2) induces accumulation of ubiquitinated proteins and exacerbates cell death in normoxic and hypoxic primary neurons. 15d-PGJ(2) covalently modifies UCH-L1 and inhibits its hydrolase activity. Pharmacologic inhibition of UCH-L1 exacerbates hypoxic neuronal death while transduction with a TAT-UCH-L1 fusion protein protects neurons from hypoxia. These studies indicate that UCH-L1 function is important in hypoxic neuronal death and that excessive production of CyPGs after stroke may exacerbate ischemic injury by modification and inhibition of UCH-L1.

Activation of the acute inflammatory response alters cytochrome P450 expression and eicosanoid metabolism.

Cytochrome P450 (P450)-mediated metabolism of arachidonic acid regulates inflammation in hepatic and extrahepatic tissue. CYP2C/CYP2J-derived epoxyeicosatrienoic and dihydroxyeicosatrienoic acids (EET+DHET) elicit anti-inflammatory effects, whereas CYP4A/CYP4F-derived 20-hydroxyeicosatetraenoic acid (20-HETE) is proinflammatory. Because the impact of inflammation on P450-mediated formation of endogenous eicosanoids is unclear, we evaluated P450 mRNA levels and P450 epoxygenase (EET+DHET) and ω-hydroxylase (20-HETE) metabolic activity in liver, kidney, lung, and heart in mice 3, 6, 24, and 48 h after intraperitoneal lipopolysaccharide (LPS) (1 mg/kg) or saline administration. Hepatic Cyp2c29, Cyp2c44, and Cyp2j5 mRNA levels and EET+DHET formation were significantly lower 24 and 48 h after LPS administration. Hepatic Cyp4a12a, Cyp4a12b, and Cyp4f13 mRNA levels and 20-HETE formation were also significantly lower at 24 h, but recovered to baseline at 48 h, resulting in a significantly higher 20-HETE/EET+DHET formation rate ratio compared with that for saline-treated mice. Renal P450 mRNA levels and P450-mediated eicosanoid metabolism were similarly suppressed 24 h after LPS treatment. Pulmonary EET+DHET formation was lower at all time points after LPS administration, whereas 20-HETE formation was suppressed in a time-dependent manner, with the lowest formation rate observed at 24 h. No differences in EET+DHET or 20-HETE formation were observed in heart. Collectively, these data demonstrate that acute activation of the innate immune response alters P450 expression and eicosanoid metabolism in mice in an isoform-, tissue-, and time-dependent manner. Further study is necessary to determine whether therapeutic restoration of the functional balance between the P450 epoxygenase and ω-hydroxylase pathways is an effective anti-inflammatory strategy.

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.

Intravenous formulation of N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine (HET0016) for inhibition of rat brain 20-hydroxyeicosatetraenoic acid formation.

N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine (HET0016) is a potent inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) formation by specific cytochrome P450 isoforms. Previous studies have demonstrated that administration of HET0016 inhibits brain formation of 20-HETE and reduces brain damage in a rat model of thromboembolic stroke. Delineation of the dose, concentration, and neuroprotective effect relationship of HET0016 has been hampered by the relative insolubility of HET0016 in aqueous solutions and the lack of information concerning the mechanism and duration of HET0016 inhibition of brain 20-HETE formation. Therefore, it was the purpose of this study to develop a water-soluble formulation of HET0016 suitable for intravenous (i.v.) administration and to determine the time course and mechanism of brain 20-HETE inhibition after in vivo dosing. In this study we report that HET0016 is a noncompetitive inhibitor of rat brain 20-HETE formation, which demonstrates a tissue concentration range for brain inhibition. In addition, we demonstrate that complexation of HET0016 with hydroxypropyl-beta-cyclodextrin results in increased aqueous solubility of HET0016 from 34.2 +/- 31.2 to 452.7 +/- 63.3 microg/ml. Administration of the complex as a single HET0016 i.v. dose (1 mg/kg) rapidly reduced rat brain 20-HETE concentrations from 289 to 91 pmol/g. Collectively, these data demonstrate that the i.v. formulation of HET0016 rapidly penetrates the rat brain and significantly inhibits 20-HETE tissue concentrations. These results will enable future studies to determine biopharmaceutics of HET0016 for inhibition of 20-HETE after cerebral ischemia.