Correlation between variant allele frequency and mean tumor molecules with tumor burden in patients with solid tumors.

Several studies have demonstrated the prognostic value of circulating tumor DNA (ctDNA); however, the correlation of mean tumor molecules (MTM)/ml of plasma and mean variant allele frequency (mVAF; %) with clinical parameters is yet to be understood. In this study, we analyzed ctDNA data in a pan-cancer cohort of 23 543 patients who had ctDNA testing performed using a personalized, tumor-informed assay (Signatera™, mPCR-NGS assay). For ctDNA-positive patients, the correlation between MTM/ml and mVAF was examined. Two subanalyses were performed: (a) to establish the association of ctDNA with tumor volume and (b) to assess the correlation between ctDNA dynamics and patient outcomes. On a global cohort, a positive correlation between MTM/ml and mVAF was observed. Among 18 426 patients with longitudinal ctDNA measurements, 13.3% had discordant trajectories between MTM/ml and mVAF at subsequent time points. In metastatic patients receiving immunotherapy (N = 51), changes in ctDNA levels expressed both in MTM/ml and mVAF showed a statistically significant association with progression-free survival; however, the correlation with MTM/ml was numerically stronger.

Intraputaminal Gene Delivery in Two Patients with Aromatic L-Amino Acid Decarboxylase Deficiency.

Background: Aromatic l-amino acid decarboxylase deficiency (AADCD) is a rare, early-onset, dyskinetic encephalopathy mostly reflecting a defective synthesis of brain dopamine and serotonin. Intracerebral gene delivery (GD) provided a significant improvement among AADCD patients (mean age, ≤6 years). Objective: We describe the clinical, biological, and imaging evolution of two AADCD patients ages >10 years after GD. Methods: Eladocagene exuparvovec, a recombinant adeno-associated virus containing the human complimentary DNA encoding the AADC enzyme, was administered into bilateral putamen by stereotactic surgery. Results: Eighteen months after GD, patients showed improvement in motor, cognitive and behavioral function, and in quality of life. Cerebral l-6-[18F] fluoro-3, 4-dihydroxyphenylalanine uptake was increased at 1 month, persisting at 1 year compared to baseline. Conclusion: Two patients with a severe form of AADCD had an objective motor and non-motor benefit from eladocagene exuparvovec injection even when treated after the age of 10 years, as in the seminal study.

A Chitosan-based Hydrogel with PLCL, ZnO NPs, and Oligoelements: A Promising Antibiotic Scaffold for Tissue Engineering / Hidrogel de Quitosano con PLCL, ZnO NPs y Oligoelementos: Un Andamio Antibacteriano Prometedor para Ingeniería de Tejidos

ABSTRACT Tissue engineering involves anchorage-dependent cells cultured on scaffolds, with growth factors added to facilitate cell proliferation. Its use in transplants implies the risk of bacterial infection. The current contribution describes the preparation and antibacterial evaluation of a chitosan-based hydrogel physically cross-linked with poly(l-lactic-coɛ-caprolactone) (PLCL) and enriched with zinc oxide nanoparticles (ZnO NPs) and trace elements (potassium and magnesium). The material was developed as a scaffold with built-in antibacterial properties. Chitosan and PLCL are biocompatible support materials applied in medicine for the repair and regeneration of damaged tissues, objectives promoted by ZnO NPs and the aforementioned trace elements. The ZnO NPs were elaborated by chemical coprecipitation. The materials were characterized by XRD, FT-IR, and SEM. Antibacterial testing was performed with strains of Escherichia coli and Staphylococcus aureus by the Kirby-Bauer method, in accordance with the NCCLS and CLSI guidelines. It was possible to obtain a homogeneous hydrogel with adequate morphology and distribution of elements. The hydrogel with 300 mM of Mg, K, and ZnO NP's showed antibacterial inhibition halos of 13 mm for S. aureus and 19 mm for E. coli. This innovative biomaterial with trace elements holds promise for tissue engineering by considering the challenge of bacterial infection.

RESUMEN La ingeniería de tejidos involucra el uso de células cultivadas en andamios con adiciones de factores de crecimiento para facilitar la proliferación celular. Su uso en trasplantes implica riesgo de infección bacteriana. La contribución actual describe la preparación y evaluación antibacteriana de un hidrogel a base de quitosano físicamente reticulado con poli (l-láctico-co-ɛ-caprolactona) (PLCL) enriquecido con nanopartículas de óxido de zinc (NP de ZnO) y oligoelementos (potasio y magnesio). El material se desarrolló como un andamio con propiedades antibacterianas. El quitosano y el PLCL son materiales de soporte biocompatibles aplicados en medicina para la reparación y regeneración de tejidos dañados, propiedades promovidas por las NP´s de ZnO y los oligoelementos antes mencionados. Las NP de ZnO se elaboraron mediante coprecipitación química. Los materiales se caracterizaron por DRX, FT-IR y SEM. Las pruebas antibacterianas se realizaron con cepas de Escherichia coli y Staphylococcus aureus por el método de KirbyBauer de acuerdo con las guías NCCLS y CLSI. Se pudo obtener un hidrogel homogéneo con adecuada morfología y distribución de elementos. El hidrogel con 300 mM de NP ZnO y oligoelementos mostró halos de inhibición antibacteriana de 13 mm para S. aureus y 19 mm para E. coli. Este biomaterial innovador con oligoelementos es prometedor para la ingeniería de tejidos al considerar el desafío de la infección bacteriana.

Further exploration of the structure-activity relationship of dual soluble epoxide hydrolase/fatty acid amide hydrolase inhibitors.

Fatty acid amide hydrolase (FAAH) is a membrane protein that hydrolyzes endocannabinoids, and its inhibition produces analgesic and anti-inflammatory effects. The soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids. EETs have anti-inflammatory and inflammation resolving properties, thus inhibition of sEH consequently reduces inflammation. Concurrent inhibition of both enzymes may represent a novel approach in the treatment of chronic pain. Drugs with multiple targets can provide a superior therapeutic effect and a decrease in side effects compared to ligands with single targets. Previously, microwave-assisted methodologies were employed to synthesize libraries of benzothiazole analogs from which high affinity dual inhibitors (e.g. 3, sEH IC50 = 9.6 nM; FAAH IC50 = 7 nM) were identified. Here, our structure-activity relationship studies revealed that the 4-phenylthiazole moiety is well tolerated by both enzymes, producing excellent inhibition potencies in the low nanomolar range (e.g. 6o, sEH IC50 = 2.5 nM; FAAH IC50 = 9.8 nM). Docking experiments show that the new class of dual inhibitors bind within the catalytic sites of both enzymes. Prediction of several pharmacokinetic/pharmacodynamic properties suggest that these new dual inhibitors are good candidates for further in vivo evaluation. Finally, dual inhibitor 3 was tested in the Formalin Test, a rat model of acute inflammatory pain. The data indicate that 3 produces antinociception against the inflammatory phase of the Formalin Test in vivo and is metabolically stable following intraperitoneal administration in male rats. Further, antinociception produced by 3 is comparable to that of ketoprofen, a traditional nonsteroidal anti-inflammatory drug. The results presented here will help toward the long-term goal of developing novel non-opioid therapeutics for pain management.

The prostaglandin pathway is activated in patients who fail medical therapy for benign prostatic hyperplasia with lower urinary tract symptoms.

BACKGROUND: Little is known about how benign prostatic hyperplasia (BPH) develops and why patients respond differently to medical therapy designed to reduce lower urinary tract symptoms (LUTS). The Medical Therapy of Prostatic Symptoms (MTOPS) trial randomized men with symptoms of BPH and followed response to medical therapy for up to 6 years. Treatment with a 5α-reductase inhibitor (5ARI) or an alpha-adrenergic receptor antagonist (α-blocker) reduced the risk of clinical progression, while men treated with combination therapy showed a 66% decrease in risk of progressive disease. However, medical therapies for BPH/LUTS are not effective in many patients. The reasons for nonresponse or loss of therapeutic response in the remaining patients over time are unknown. A better understanding of why patients fail to respond to medical therapy may have a major impact on developing new approaches for the medical treatment of BPH/LUTS. Prostaglandins (PG) act on G-protein-coupled receptors (GPCRs), where PGE2 and PGF2 elicit smooth muscle contraction. Therefore, we measured PG levels in the prostate tissue of BPH/LUTS patients to assess the possibility that this signaling pathway might explain the failure of medical therapy in BPH/LUTS patients. METHOD: Surgical BPH (S-BPH) was defined as benign prostatic tissue collected from the transition zone (TZ) of patients who failed medical therapy and underwent surgical intervention to relieve LUTS. Control tissue was termed Incidental BPH (I-BPH). I-BPH was TZ obtained from men undergoing radical prostatectomy for low-volume, low-grade prostatic adenocarcinoma (PCa, Gleason score ≤ 7) confined to the peripheral zone. All TZ tissue was confirmed to be cancer-free. S-BPH patients divided into four subgroups: patients on α-blockers alone, 5ARI alone, combination therapy (α-blockers plus 5ARI), or no medical therapy (none) before surgical resection. I-BPH tissue was subgrouped by prior therapy (either on α-blockers or without prior medical therapy before prostatectomy). We measured prostatic tissue levels of prostaglandins (PGF2α , PGI2 , PGE2 , PGD2 , and TxA2 ), quantitative polymerase chain reaction levels of mRNAs encoding enzymes within the PG synthesis pathway, cellular distribution of COX1 (PTGS1) and COX2 (PTGS2), and tested the ability of PGs to contract bladder smooth muscle in an in vitro assay. RESULTS: All PGs were significantly elevated in TZ tissues from S-BPH patients (n = 36) compared to I-BPH patients (n = 15), regardless of the treatment subgroups. In S-BPH versus I-BPH, mRNA for PG synthetic enzymes COX1 and COX2 were significantly elevated. In addition, mRNA for enzymes that convert the precursor PGH2 to metabolite PGs were variable: PTGIS (which generates PGI2 ) and PTGDS (PGD2 ) were significantly elevated; nonsignificant increases were observed for PTGES (PGE2 ), AKR1C3 (PGF2α ), and TBxAS1 (TxA2 ). Within the I-BPH group, men responding to α-blockers for symptoms of BPH but requiring prostatectomy for PCa did not show elevated levels of COX1, COX2, or PGs. By immunohistochemistry, COX1 was predominantly observed in the prostatic stroma while COX2 was present in scattered luminal cells of isolated prostatic glands in S-BPH. PGE2 and PGF2α induced contraction of bladder smooth muscle in an in vitro assay. Furthermore, using the smooth muscle assay, we demonstrated that α-blockers that inhibit alpha-adrenergic receptors do not appear to inhibit PG stimulation of GPCRs in bladder muscle. Only patients who required surgery to relieve BPH/LUTS symptoms showed significantly increased tissue levels of PGs and the PG synthetic enzymes. CONCLUSIONS: Treatment of BPH/LUTS by inhibition of alpha-adrenergic receptors with pharmaceutical α-blockers or inhibiting androgenesis with 5ARI may fail because of elevated paracrine signaling by prostatic PGs that can cause smooth muscle contraction. In contrast to patients who fail medical therapy for BPH/LUTS, control I-BPH patients do not show the same evidence of elevated PG pathway signaling. Elevation of the PG pathway may explain, in part, why the risk of clinical progression in the MTOPS study was only reduced by 34% with α-blocker treatment.

Topical Sensor for the Assessment of PET Dose Administration: Metric Performance with an Autoinjector.

Extravasation or partial extravasation of the radiopharmaceutical dose in PET can undermine SUV and image quality. A topical sensor has been validated using several metrics to characterize injection quality after manual injection. The performance of these metrics for autoinjector administration has been assessed. Methods: A single PET/CT scanner at a single site was used to characterize injections using an autoinjector with standardized apparatus, flush volume, and infusion rate (1-min infusion followed by 2 syringe flushes) for 18F-FDG, 68Ga-prostate-specific membrane antigen, and 68Ga-DOTATATE. In total, 296 patients with topical application of sensors were retrospectively analyzed using conventional statistical analysis and an artificial neural network. Results: Partial extravasation was noted in 1.3% of studies, with 9.1% (inclusive of partial extravasation) identified to have an injection anomaly (e.g., venous retention). Extravasation was independently predicted by the time that elapsed as the counts recorded by the injection sensor fell from the maximum value to within 200% of the reference sensor counts greater than 1,200 s; as the difference in counts for injection and reference sensors, normalized by dose, from 4 min after injection greater than 25; and as the ratio of the average counts per second recorded by the injection sensor at the end of a monitoring period to those of the reference sensor greater than 2. Conclusion: Extravasation and partial extravasation of PET doses are readily detected and differentiated using time-activity curve metrics. The metrics can provide the insight that could inform image quality or SUV accuracy issues. Further validation of key metrics is recommended in a larger and more diverse cohort.

Primary Resistance to PD-1-Based Immunotherapy-A Study in 319 Patients with Stage IV Melanoma.

BACKGROUND: Primary resistance to immunotherapy can be observed in approximately 40-65% of the stage IV melanoma patients treated with immune checkpoint inhibitors. A minority of the patients receive a second-line therapy, and the clinical benefit is small. PATIENTS AND METHODS: Stage IV melanoma patients treated with first-line PD-1-based immunotherapy between January 2015 and December 2018 were investigated. Primary resistance was defined as progressive disease (PD) at the time of the first tumor assessment after starting immunotherapy. Patients with complete response, partial response, and stable disease were classified as having disease control (DC). Overall survival (OS) and progression-free survival (PFS) were evaluated by Kaplan-Meier estimator. Univariate and multivariate logistic regression analyses were performed to determine prognostic factors associated with OS. RESULTS: Three hundred and nineteen patients were included, and 40% had primary resistance to immunotherapy. The median follow-up time was 22 months. Patients with primary resistance had 1-, 2-, and 3-year OS rates of 41%, 15%, and 10%, respectively, compared to 91%, 81%, and 65% for the patients who achieved DC. The following independently significant prognostic factors for OS were identified: protein S100B level and primary tumor localization. There was a statistically significant difference for OS (p < 0.0001) but not for PFS (p = 0.230) when analyzing risk groups formed with a combination of these two variables (low-, intermediate-, and high-risk subgroups). CONCLUSIONS: Melanoma patients with primary resistance to immunotherapy have a dismal prognosis. Response at the first tumor assessment after starting immunotherapy is a stronger prognostic factor for the further course of the disease than pretreatment risk factors.

Detection of 18F-FDG Dose Leakage Using a Topical Device.

PET and SUV depend on reliable pharmacokinetic modeling, part of which is predictable dose delivery. Partial extravasation of the intravenous dose administered undermines the predictability of dose delivery and, potentially, the accuracy of the SUV calculation. Use of the Lara device with topical sensors is a simple, noninvasive way to determine partial dose extravasation. As part of routine monitoring of 18F-FDG PET administrations, an interesting case was identified that mimicked extravasation but represented dose leakage during infusion via an automatic injector. The Lara device provided a useful tool for more timely critical evaluation and problem solving, extending advantages to the patient and practice.

Coaching primary care clinics for HPV vaccination quality improvement: Comparing in-person and webinar implementation.

State health departments commonly use quality improvement coaching as an implementation strategy for improving low human papillomavirus (HPV) vaccination coverage, but such coaching can be resource intensive. To explore opportunities for improving efficiency, we compared in-person and webinar delivery of coaching sessions on implementation outcomes, including reach, acceptability, and delivery cost. In 2015, we randomly assigned 148 high-volume primary care clinics in Illinois, Michigan, and Washington State to receive either in-person or webinar coaching. Coaching sessions lasted about 1 hr and used our Immunization Report Card to facilitate assessment and feedback. Clinics served over 213,000 patients ages 11-17. We used provider surveys and delivery cost assessment to collect implementation data. This report is focused exclusively on the implementation aspects of the intervention. More providers attended in-person than webinar coaching sessions (mean 9 vs. 5 providers per clinic, respectively, p = .004). More providers shared the Immunization Report Card at clinic staff meetings in the in-person than webinar arm (49% vs. 20%; p = .029). In both arms, providers' belief that their clinics' HPV vaccination coverage was too low increased, as did their self-efficacy to help their clinics improve (p < .05). Providers rated coaching sessions in the two arms equally highly on acceptability. Delivery cost per clinic was $733 for in-person coaching versus $461 for webinar coaching. In-person and webinar coaching were well received and yielded improvements in provider beliefs and self-efficacy regarding HPV vaccine quality improvement. In summary, in-person coaching cost more than webinar coaching per clinic reached, but reached more providers. Further implementation research is needed to understand how and for whom webinar coaching may be appropriate.

N-acetylcysteine targets 5 lipoxygenase-derived, toxic lipids and can synergize with prostaglandin E2 to inhibit ferroptosis and improve outcomes following hemorrhagic stroke in mice.

OBJECTIVES: N-acetylcysteine (NAC) is a clinically approved thiol-containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an "antioxidant," poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke. METHODS: Hemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5-lipoxygenase (ALOX5) knockout mice, and viral-gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC. RESULTS: NAC prevented hemin-induced ferroptosis by neutralizing toxic lipids generated by arachidonate-dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione-dependent enzymes such as glutathione S-transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E2 (PGE2 ). INTERPRETATION: NAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE2 in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854-872.

Validation of an electronic program for pathologist training in the interpretation of a complex companion diagnostic immunohistochemical assay.

Companion diagnostics assay interpretation can select patients with the greatest targeted therapy benefits. We present the results from a prospective study demonstrating that pathologists can effectively learn immunohistochemical assay-interpretation skills from digital image-based electronic training (e-training). In this study, e-training was used to train board-certified pathologists to evaluate non-small cell lung carcinoma for eligibility for treatment with onartuzumab, a MET-inhibiting agent. The training program mimicked the live training that was previously validated in clinical trials for onartuzumab. A digital interface was developed for pathologists to review high-resolution, static images of stained slides. Sixty-four pathologists practicing in the United States enrolled while blinded to the type of training. After training, both groups completed a mandatory final test using glass slides. The results indicated both training modalities to be effective. Overall, 80.6% of e-trainees and 72.7% of live trainees achieved passing scores (at least 85%) on the final test. All study participants reported that their training experience was "good" and that they had received sufficient information to determine the adequacy of case slide staining to score each case. This study established that an e-training program conducted under highly controlled conditions can provide pathologists with the skills necessary to interpret a complex assay and that these skills can be equivalent to those achieved with face-to-face training using conventional microscopy. Programs of this type are scalable for global distribution and offer pathologists the potential for readily accessible and robust training in new companion diagnostic assays linked to novel, targeted, adjuvant therapies for cancer patients.

Measurement of F2- isoprostanes and isofurans using gas chromatography-mass spectrometry.

F2-Isoprostanes (IsoPs) are isomers of prostaglandin F2α formed from the nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Since discovery of these molecules by Morrow and Roberts in 1990, F2-IsoPs have been shown to be excellent biomarkers as well as potent mediators of oxidative stress in vivo in humans. Isofurans (IsoFs) are also oxidation products generated from the nonenzymatic oxidation of arachidonic acid. IsoFs are preferentially formed instead of F2-IsoPs in settings of increased oxygen tension. The protocol presented herein is the current methodology that our laboratory uses to quantify F2-IsoPs and IsoFs in biological tissues and fluids using gas chromatography/mass spectrometry (GC/MS). A variety of analytical procedures to measure F2-IsoPs, including other GC/MS methods and liquid chromatography/MS and immunological approaches, are reported in the literature. This method provides a very low limit of quantitation and is suitable for analysis of both F2-IsoPs and IsoFs from a variety of biological sources including urine, plasma, tissues, cerebral spinal fluid, exhaled breath condensate, and amniotic fluid, among others.

Amniotic fluid eicosanoids in preterm and term births: effects of risk factors for spontaneous preterm labor.

OBJECTIVE: To evaluate amniotic fluid arachidonic acid metabolites using enzymatic and nonenzymatic (lipid peroxidation) pathways in spontaneous preterm birth and term births, and to estimate whether prostanoid concentrations correlate with risk factors (race, cigarette smoking, and microbial invasion of amniotic cavity) associated with preterm birth. METHODS: In a case-control study, amniotic fluid was collected at the time of labor or during cesarean delivery. Amniotic fluid samples were subjected to gas chromatography, negative ion chemical ionization, and mass spectrometry for prostaglandin (PG) E2, PGF2α, and PGD2 and for 6-keto-PGF1α (thromboxane 2 and F2-isoprostane). Primary analysis examined differences between prostanoid concentrations in preterm birth (n=133) compared with term births (n=189). Secondary stratified analyses (by race, cigarette smoking, and microbial invasion of amniotic cavity) compared eicosanoid concentrations in three epidemiological risk factors. RESULTS: Amniotic fluid F2-isoprostane, PGE2, and PGD2 were significantly higher at term than in preterm birth, whereas PGF2α was higher in preterm birth 6-keto-PGF1α and thromboxane 2 concentrations were not different. Data stratified by race (African American or white) showed no significant disparity among prostanoid concentrations. Regardless of gestational age status, F2-isoprostane was threefold higher in smokers, and other eicosanoids were also higher in smokers compared with nonsmokers. Preterm birth with microbial invasion of amniotic cavity had significantly higher F2-isoprostane compared with preterm birth without microbial invasion of amniotic cavity. CONCLUSION: Most amniotic fluid eicosanoid concentrations (F2-isoprostane, PGE2, and PGD2), are higher at term than in preterm births. The only amniotic fluid eicosanoid that is not higher at term is PGF2α.

Intra-person variation of urinary biomarkers of oxidative stress and inflammation.

BACKGROUND: Oxidative stress and inflammation have been linked to many chronic diseases including cancer and cardiovascular diseases. Urinary levels of F(2)-isoprostanes (F(2)-IsoPs), 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (15-F(2t)-IsoP-M), a major metabolite of F(2)-IsoPs, prostaglandin E(2) metabolite (PGE-M), and leukotriene E(4) (LTE(4)) have been proposed as biomarkers for oxidative stress and inflammation. However, little information is available regarding the intra-person variation of these biomarkers, hindering their application in epidemiologic studies. METHODS: We evaluated the intra-person variation of these four urinary biomarkers among 48 randomly chosen participants of a validation study of a population-based cohort, the Shanghai Men's Health Study. Four spot urine samples, collected during each season over a 1-year period, were measured for these biomarkers. RESULTS: The intraclass correlation coefficients for F(2)-IsoPs, 15-F(2t)-IsoP-M, PGE-M, and LTE(4) were 0.69, 0.76, 0.67, and 0.64, respectively. The Spearman correlation coefficients, derived by using bootstrap analysis of single spot measurements and the average of the other three seasonal measurements, were 0.47, 0.60, 0.61, and 0.57 for F(2)-IsoPs, 15-F(2t)-IsoP-M, PGE-M, and LTE(4). Except for high correlations between F(2)-IsoPs and 15-F(2t)-IsoP-M (r = 0.65), the other biomarkers were moderately correlated (r = 0.21-0.44). CONCLUSIONS: Our study results suggest that these four urinary biomarkers have relatively low intra-person variation over a 1-year period. IMPACT: Spot measurements of F(2)-IsoPs, 15-F(2t)-IsoP-M, PGE-M, and LTE(4) could be useful as biomarkers of oxidative stress and inflammation status for epidemiologic studies.

Formation of highly reactive cyclopentenone isoprostane compounds (A3/J3-isoprostanes) in vivo from eicosapentaenoic acid.

Omega-3 (omega-3) polyunsaturated fatty acids (PUFAs) found in marine fish oils are known to suppress inflammation associated with a wide variety of diseases. Eicosapentaenoic acid (EPA) is one of the most abundant omega-3 fatty acids in fish oil, but the mechanism(s) by which EPA exerts its beneficial effects is unknown. Recent studies, however, have demonstrated that oxidized EPA, rather than native EPA, possesses anti-atherosclerotic, anti-inflammatory, and anti-proliferative effects. Very few studies to date have investigated which EPA oxidation products are responsible for this bioactivity. Our research group has previously reported that anti-inflammatory prostaglandin A(2)-like and prostaglandin J(2)-like compounds, termed A(2)/J(2)-isoprostanes (IsoPs), are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid and represent one of the major products resulting from the oxidation of this PUFA. Based on these observations, we questioned whether cyclopentenone-IsoP compounds are formed from the oxidation of EPA in vivo. Herein, we report the formation of cyclopentenone-IsoP molecules, termed A(3)/J(3)-IsoPs, formed in abundance in vitro and in vivo from EPA peroxidation. Chemical approaches coupled with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) were used to structurally characterize these compounds as A(3)/J(3)-IsoPs. We found that levels of these molecules increase approximately 200-fold with oxidation of EPA in vitro from a basal level of 0.8 +/- 0.4 ng/mg EPA to 196 +/- 23 ng/mg EPA after 36 h. We also detected these compounds in significant amounts in fresh liver tissue from EPA-fed rats at basal levels of 19 +/- 2 ng/g tissue. Amounts increased to 102 +/- 15 ng/g tissue in vivo in settings of oxidative stress. These studies have, for the first time, definitively characterized novel, highly reactive A/J-ring IsoP compounds that form in abundance from the oxidation of EPA in vivo.

Quantification of the major urinary metabolite of PGE2 by a liquid chromatographic/mass spectrometric assay: determination of cyclooxygenase-specific PGE2 synthesis in healthy humans and those with lung cancer.

Prostaglandin (PG)E2 is a major cyclooxygenase (COX) product that is important in human physiology and pathophysiology. Quantification of systemic PG production in humans is best assessed by measuring excreted urinary metabolites. Accurate and easy-to-perform assays to quantify the major urinary metabolite of PGE2, 11alpha-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), do not exist. We now report the development of a robust and facile method to measure urinary PGE-M excretion in humans using stable isotope dilution techniques employing liquid chromatography/tandem mass spectrometry (LC/MS/MS). Concentrations of the metabolite in urine from healthy humans are nearly twofold greater in men than in women (10.4+/-1.5 vs. 6.0+/-0.7 ng/mg creatinine). Levels of PGE-M in healthy humans are suppressed significantly not only by the nonselective COX inhibitor ibuprofen but also by the COX-2 selective inhibitor rofecoxib, suggesting that the majority of PGE2 formed in vivo is derived from COX-2. Increased COX-2 expression and increased PGE2 production are associated with malignancy. Levels of PGE-M were found to be greatly increased in humans with unresectable non-small cell cancer of the lung, and this increase is dramatically reduced by administration of the COX-2 inhibitor celecoxib, implying that COX-2 contributes significantly to the overproduction of PGE2. In summary, quantification of PGE-M using LC/MS/MS provides a facile and accurate method to assess PGE2 formation in human physiological and pathophysiological processes.