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1.  Centralized Oversight of Physician–Scientist Faculty Development at Vanderbilt: Early Outcomes 
Purpose
In 2000, faced with a national concern over the decreasing number of physician–scientists, Vanderbilt School of Medicine established the institutionally funded Vanderbilt Physician–Scientist Development (VPSD) program to provide centralized oversight and financial support for physician–scientist career development. In 2002, Vanderbilt developed the National Institutes of Health (NIH)-funded Vanderbilt Clinical Research Scholars (VCRS) program using a similar model of centralized oversight. The authors evaluate the impact of the VPSD and VCRS programs on early career outcomes of physician–scientists.
Method
Physician–scientists who entered the VPSD or VCRS programs from 2000 through 2006 were compared with Vanderbilt physician–scientists who received NIH career development funding during the same period without participating in the VPSD or VCRS programs.
Results
Seventy-five percent of VPSD and 60% of VCRS participants achieved individual career award funding at a younger age than the comparison cohort. This shift to career development award funding at a younger age among VPSD and VCRS scholars was accompanied by a 2.6-fold increase in the number of new K awards funded and a rate of growth in K-award dollars at Vanderbilt that outpaced the national rate of growth in K-award funding.
Conclusions
Analysis of the early outcomes of the VPSD and VCRS programs suggests that centralized oversight can catalyze growth in the number of funded physician–scientists at an institution. Investment in this model of career development for physician–scientists may have had an additive effect on the recruitment and retention of talented trainees and junior faculty.
doi:10.1097/ACM.0b013e3181850950
PMCID: PMC3780596  PMID: 18820531
2.  Sex differences in urinary biomarkers of vascular and endothelial function in HIV-infected persons receiving antiretroviral therapy 
Antiviral therapy  2011;17(3):485-493.
Background
Cardiovascular disease (CVD) risk can be underestimated in HIV-infected patients receiving antiretroviral therapy (ART). Novel CVD risk markers in this population are needed. We hypothesized that eicosanoid metabolite production is increased with metabolic complications of ART. Our objective was to determine relationships between urine eicosanoids and traditional CVD risk factors in a cohort of HIV-infected persons receiving ART.
Methods
Cross-sectional analysis of 107 individuals from a prospective cohort study with urine eicosanoids (isoprostane [15-F2t-IsoP], prostaglandin-E metabolite [PGE-M], thromboxane metabolite [11dTxB2], prostacyclin metabolite [PGI-M]) determined by gas or liquid chromatography-mass spectrometry.
Results
15-F2t-IsoP was higher (p=0.003), 11dTxB2 tended to be higher (p=0.07), and PGE-M was lower (p=0.003) in females than in males. The overall median Framingham score was 4 (IQR 1 – 7). In multivariable analyses adjusting for age, CD4+ T-cells, smoking status, nonsteroidal anti-inflammatory use, aspirin use, and body mass index (BMI), associations included: higher 15-F2t-IsoP with female sex (p=0.004) and current smoking (p=0.04), lower PGE-M with female sex (p=0.005) and higher BMI (p=0.03), higher 11dTxB2 with increasing age (p=0.02) and current smoking (p=0.04), lower 11dTxB2 with higher BMI (p=0.02), and higher PGI-M with current smoking (p=0.04).
Conclusions
In this pilot study of predominantly virologically suppressed HIV-infected individuals on ART, there were sex-specific differences in urinary eicosanoids, with females having more risk-associated parameters despite low Framingham score. Eicosanoids might be useful CVD biomarkers in ART-treated, HIV-infected patients. Future studies should examine eicosanoids while assessing effects of specific ART regimens and targeted interventions on CVD outcomes.
doi:10.3851/IMP1990
PMCID: PMC3766979  PMID: 22293574
3.  Cyclopentenone Eicosanoids as Mediators of Neurodegeneration: A Pathogenic Mechanism of Oxidative Stress-Mediated and Cyclooxygenase-Mediated Neurotoxicity 
The activation of cyclooxygenase enzymes in the brain has been implicated in the pathogenesis of numerous neurodegenerative conditions. Similarly, oxidative stress is believed to be a major contributor to many forms of neurodegeneration. These 2 distinct processes are united by a common characteristic: the generation of electrophilic cyclopentenone eicosanoids. These cyclopentenone compounds are defined structurally by the presence of an unsaturated carbonyl moiety in their prostane ring, and readily form Michael adducts with cellular thiols, including those found in glutathione and proteins. The cyclopentenone prostaglandins (PGs) PGA2, PGJ2, and 15-deoxy-Δ12,14 PGJ2, enzymatic products of cyclooxygenase-mediated arachidonic acid metabolism, exert a complex array of potent neurodegenerative, neuroprotective, and anti-inflammatory effects. Cyclopentenone isoprostanes (A2/J2-IsoPs), products of non-enzymatic, free radical-mediated arachidonate oxidation, are also highly bioactive, and can exert direct neurodegenerative effects. In addition, cyclopentenone products of docosahexaenoic acid oxidation (cyclopentenone neuroprostanes) are also formed abundantly in the brain. For the first time, the formation and biological actions of these various classes of reactive cyclopentenone eicosanoids are reviewed, with emphasis on their potential roles in neurodegeneration. The accumulating evidence suggests that the formation of cyclopentenone eicosanoids in the brain may represent a novel pathogenic mechanism, which contributes to many neurodegenerative conditions.
PMCID: PMC2881556  PMID: 15912888
4.  Comparison of Three Oxidative Stress Biomarkers in a Sample of Healthy Adults 
Biomarkers  2009;14(8):587-595.
Oxidative stress is a potentially important etiologic factor for many chronic diseases, including cardiovascular disease, neurodegenerative disease, and cancer, yet studies often find inconsistent results. The associations between three of the most widely-used biomarkers of oxidative stress, i.e., F2-isoprostanes for lipid peroxidation and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) and the comet assay with FPG for oxidative DNA damage, were compared in a sample of 135 healthy African American and White adults. Modest associations were observed between F2-isoprostanes and the comet assay (r=0.22, p=0.01), but there were no significant correlations between 8-oxo-dG and the comet assay (r=−0.09) or F2-IsoP (r=−0.04). These results are informative for researchers seeking to compare results pertaining to oxidative stress across studies and/or assessment methods in healthy disease-free populations. The development and use of oxidative stress biomarkers is a promising field; however, additional validation studies are necessary to establish accuracy and comparability across oxidative stress biomarkers.
doi:10.3109/13547500903183954
PMCID: PMC3290341  PMID: 20001708
Oxidation DNA damage; lipid peroxidation; 8-hydroxy-2-deoxyguanosine; comet assay; isoprostanes
5.  p66shc's role as an essential mitophaghic molecule in controlling neuronal redox and energetic tone 
Autophagy  2010;6(7):948-949.
Stroke is the leading cause of adult disability in the U.S. and is now recognized as a global epidemic. There are currently no FDA-approved drugs to block the cell death that results from oxygen and glucose deprivation. This void in clinical medicine has sparked an intense interest in understanding endogenous cellular protective pathways that might be exploited for therapeutic development. The work highlighted here describes the critical role between redox tone and energetic stress signaling in mediating mitophagy and determining neuronal cell fate following acute oxygen glucose deprivation.
doi:10.4161/auto.6.7.13007
PMCID: PMC3679093  PMID: 20724835
hypoxia; ischemia; mitochondria; mitophagy; preconditioning; neurodegeneration
6.  Highly-sensitive c-reactive protein, body mass index, and serum lipids in HIV-infected persons receiving antiretroviral therapy: a longitudinal study 
Background
Antiretroviral therapy (ART) affects cardiovascular disease (CVD) risk. In the general population, hsCRP is an established predictor of future coronary events. Little is known about its utility in chronic inflammatory conditions such as HIV-infection. We assessed relationships between hsCRP and metabolic parameters over time in HIV-infected patients on ART.
Methods
Data are from a prospective cohort of HIV-infected adults enrolled June 2005-July 2007. Participants were receiving ART, had HIV-1 RNA <10,000 copies/mL, and no diabetes or CVD. Non-linear mixed effect regression models assessed relationships between body mass index (BMI), lipids, and hsCRP over time adjusting for covariates.
Results
94 individuals had data from ≥ 1 study visit. Median age was 44 years, 27% were female, 57% white, and 54% were on protease inhibitors. Median CD4+ T-cells, HIV-1 RNA, and hsCRP were 502 cells/mm3, 50 copies/mL, and 2.94 mg/L, respectively. Median Framingham score was 3. Multivariate analysis identified associations between increased hsCRP and greater BMI (p=0.001), higher non-HDL-cholesterol (p=0.013) and triglycerides (p=0.017), and lower HDL-cholesterol (p=0.015).
Conclusions
Among HIV-infected adults with low estimated CVD risk and virologic suppression on ART, hsCRP was elevated and independently associated with BMI and lipid changes. Future studies should assess associations between hsCRP and clinical outcomes.
PMCID: PMC2794651  PMID: 19911471
HIV; metabolic complications; hsCRP; body mass index; lipids
7.  Restoration of On-Time Embryo Implantation Corrects the Timing of Parturition in Cytosolic Phospholipase A2 Group IVA Deficient Mice1 
Biology of Reproduction  2009;81(6):1131-1138.
Cytosolic phospholipase A2 (cPLA2, PLA2G4A) catalyzes the release of arachidonic acid for prostaglandin synthesis by cyclooxygenase 1 (PTGS1) and cyclooxygenase 2 (PTGS2). Mice with Pla2g4a deficiency have parturition delay and other reproductive deficits, including deferred onset of implantation, crowding of implantation sites, and small litters. In this study, we examined the contribution of PLA2G4A to parturition in mice. Pla2g4a mRNA and protein expression were discretely localized in the term and preterm uterine luminal epithelium and colocalized with Ptgs1, but not Ptgs2, expression. The levels of PGE2, PGF2alpha, 6-keto-PGF1alpha, and TxB2 were significantly decreased in Pla2g4a-null uterine tissues, similar to Ptgs1-null uteri, consistent with predominance of PLA2G4A-PTGS1-mediated prostaglandin synthesis in preparation for murine parturition. Litter size was strongly associated with the timing of parturition in Pla2g4a-null mice but could not fully account for the parturition delay. Pla2g4a-null females that received PGE2 + carbaprostacyclin at the time of implantation delivered earlier (20.5 ± 0.2 days vs. 21.6 ± 0.2 days, P < 0.01), although litter size was not improved (4.6 vs. 4.4 pups per litter, P = 0.6). After correction for small litter size, multivariate analysis indicated that Pla2g4a-null mice given prostaglandin treatment to improve implantation timing had gestational length that was similar to wild-type and Pla2g4a heterozygous mice. These results indicate that, despite specific Pla2g4a expression and function in term gestation uteri, the delayed parturition phenotype in Pla2g4a-null mice is primarily due to deferral of implantation. The role of PLA2G4A in timely parturition appears to be critically related to its actions in early pregnancy.
The delayed parturition phenotype in Pla2g4a-null mice is primarily due to deferral of implantation. The role of PLA2G4A in timely parturition appears to be critically related to its actions in early pregnancy.
doi:10.1095/biolreprod.109.079061
PMCID: PMC2802236  PMID: 19684335
cPLA2; mouse; parturition; phospholipase; PLA2G4A; pregnancy; prostaglandin; uterus
8.  Neurotoxic lipid peroxidation species formed by ischemic stroke increase injury 
Free radical biology & medicine  2009;47(10):1422-1431.
Stroke is the third leading cause of death in the United States yet no neuroprotective agents for treatment are clinically available. There is a pressing need to understand the signaling molecules which mediate ischemic cell death and identify novel neuroprotective targets. Cyclopentenone isoprostanes (IsoP), formed following free radical mediated peroxidation of arachidonic acid, are used as markers of stress but their bioactivity is poorly understood. We have recently shown that 15-A2t-Isop is a potent neurotoxin in vitro and increases the free radical burden in neurons. In this work, we demonstrate that 15-A2t-IsoP is abundantly produced in stroke infarcted human cortical tissue. Using primary neuronal cultures we found that minimally toxic exposure to 15-A2t-IsoP does not alter ATP content, but in combination with oxygen glucose deprivation resulted in a significant hyperpolarization of the mitochondrial membrane and dramatically increased neuronal cell death. In the presence of Ca2+, 15-A2t-IsoP led to a rapid induction of the permeability transition pore and release of cytochrome c. Taken with our previous work, these data support a model in which ischemia causes generation of reactive oxygen species, calcium influx, lipid peroxidation and 15-A2t-IsoP formation. These factors combine to enhance opening of the permeability transition pore leading to cell death subsequent to mitochondrial cytochrome c release. This data is the first documentation of significant 15-A2t-IsoP formation following acute ischemic stroke and suggests addition of 15-A2t-IsoP to in vitro models of ischemia may help to more fully recapitulate stroke injury.
doi:10.1016/j.freeradbiomed.2009.08.011
PMCID: PMC2767385  PMID: 19699297
9.  Elevated Levels of Urinary Prostaglandin E Metabolite Indicate a Poor Prognosis in Ever Smoker Head and Neck Squamous Cell Carcinoma Patients 
Cyclooxygenase (COX)-derived prostaglandin E2 (PGE2) plays a role in the development and progression of several tumor types including head and neck squamous cell carcinoma (HNSCC). Measurements of urinary PGE metabolite (PGE-M) can be used as an index of systemic PGE2 production. In ever smokers, increased levels of urinary PGE-M reflect increased COX-2 activity. In this study, we determined whether baseline levels of urinary PGE-M were prognostic for ever smoker HNSCC patients. A retrospective chart review of ever smoker HNSCC patients treated with curative intent was performed. Fifteen of 31 evaluable patients developed progressive disease (recurrence or a second primary tumor) after a median follow-up of 38 months. There were no statistically significant differences between patients with (n=15) or without disease progression (n=16) with regard to stage, site, treatment received, smoking status and aspirin use during follow-up. Median urinary PGE-M levels were significantly higher in HNSCC patients with disease progression (21.7 ng/mg creatinine) compared with patients without (13.35 ng/mg creatinine), P=0.03. Importantly, patients with high baseline levels of urinary PGE-M had a significantly greater risk of disease progression (HR=4.76, 95% CI= (1.31, 17.30), P<0.01) and death (HR=9.54, 95% CI= (1.17, 77.7), P=0.01) than patients with low baseline levels of urinary PGE-M. These differences were most evident among patients with early stage disease. Taken together, our findings suggest that high baseline levels of urinary PGE-M indicate a poor prognosis in HNSCC patients. Possibly, HNSCC patients with high COX-2 activity manifested by elevated urinary PGE-M will benefit from treatment with a COX-2 inhibitor.
doi:10.1158/1940-6207.CAPR-09-0093
PMCID: PMC2784194  PMID: 19843689
Smoking; biomarker; HNSCC; prognosis; prostaglandin
10.  Quantification of F2-Isoprostanes as a Reliable Index of Oxidative Stress in vivo Using Gas Chromatography - Mass Spectrometry (GC-MS) Method 
Free radical biology & medicine  2009;47(8):1101-1107.
Free radical-induced lipid peroxidation has been implicated in a number of human diseases including atherosclerosis, cancer, and neurodegenerative diseases. F2-Isoprostanes (IsoPs) are isomers of prostaglandin PGF2α that are generated in vivo from the free radical-initiated peroxidation of arachidonic acid independent of cyclooxygenase enzymes. Since the discovery of the IsoPs in the early 1990s, a large body of evidence has been accumulated to indicate that quantification of these F2-IsoPs represents the most reliable biomarker to assess oxidative stress in vivo. A variety of analytical approaches have been developed for the quantification of these novel compounds; these methods include mass spectrometry (MS) detection coupled to gas chromatography (GC) or liquid chromatography (LC) separation, and detection using immunological approaches. This article summarizes our current methodology to quantify F2-IsoPs in biological fluids and tissues using GC-MS. This method includes solid phase extraction (SPE), thin layer chromatography (TLC) purification, chemical derivatization, and MS detection using negative ion chemical ionization (NICI) coupled with GC. The protocol described herein has been optimized and validated to provide the best sensitivity and selectivity for quantification of F2-IsoPs from a variety of biological sources.
doi:10.1016/j.freeradbiomed.2009.07.028
PMCID: PMC2749920  PMID: 19647073
11.  Essential Role of the Redox Sensitive Kinase p66shc in Determining Energetic and Oxidative Status and Cell Fate in Neuronal Preconditioning 
Ischemic preconditioning is a phenomenon in which low level stressful stimuli upregulate endogenous defensive programs resulting in subsequent resistance to otherwise lethal injuries. We previously observed that signal transduction systems typically associated with neurodegeneration such as caspase activation are requisite events for the expression of tolerance and induction of HSP70. In this work, we sought to determine the extent and duration of oxidative and energetic dysfunction as well as the role of effector kinases on metabolic function in preconditioned cells. Using an in vitro neuronal culture model, we observed a robust increase in Raf and p66Shc activation within 1h of preconditioning. Total ATP content decreased by 25% 3h after preconditioning but returned to baseline by 24h. Use of a free radical spin trap or p66shc inhibitor increased ATP content whereas a Raf inhibitor had no effect. Phosphorylated p66shc rapidly relocalized to the mitochondria and in the absence of activated p66shc, autophagic processing increased. The constitutively expressed chaperone HSC70 relocalized to autophagosomes. Preconditioned cells experience significant total oxidative stress measured by F2-isoprostanes and neuronal stress evaluated by F4-neuroprostane measurement. Neuroprostane levels were enhanced in the presence of Shc inhibitors. Finally, we found that inhibiting either p66shc or Raf blocked neuroprotection afforded by preconditioning as well as upregulation of HSP70 suggesting both kinases. This is the first work to demonstrate the essential role of p66shc in mediating requisite mitochondrial and energetic compensation following preconditioning and suggests a mechanism by which protein and organelle damage mediated by ROS can increase HSP70.
doi:10.1523/JNEUROSCI.6366-09.2010
PMCID: PMC2869196  PMID: 20392947
excitotoxicity; preconditioning; tolerance; ischemia; p66Shc; Raf kinase; heat shock protein 70; apoptosis; autophagy; extracellular regulated kinase; p38
12.  Dietary Supplementation of ω-3 Fatty Acid - Containing Fish Oil Suppresses F2- Isoprostanes But Enhances Inflammatory Cytokine Response in a Mouse Model of Ovalbumin-Induced Allergic Lung Inflammation 
Free radical biology & medicine  2009;47(5):622-628.
Epidemiological and clinical evidence has suggested that increased dietary intake of fish oil containing w-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may be associated with a reduced risk of asthma. However, interventional studies on these effects have been equivocal and controversial. Free radical oxidation products of lipids and cyclooxygenases-derived prostaglandins are believed to play an important role in asthma, and fish oil supplementation may modulate the levels of these critical lipid mediators. We employed a murine model of allergic inflammation produced by sensitization to ovalbumin (OVA) to study the effects of fish oil supplementation on airway inflammation. Our studies demonstrated that ω-3 fatty acids were dose-dependently incorporated into mouse lung tissue after dietary supplementation. We examined the oxidative stress status by measuring the levels of isoprostanes (IsoPs), the gold standard for oxidative stress in vivo. OVA challenge caused significant increase of F2-IsoPs in mouse lung, suggesting an elevated level of oxidative stress. Comparing to the control group, fish oil supplementation led to a significant reduction of F2-IsoP (from arachidonic acid) with a concomitant increase of F3-IsoPs (from EPA) and F4-IsoPs (from DHA). Surprisingly, however, fish oil supplementation enhanced production of pro-inflammatory cytokine IL-5 and IL-13. Furthermore, fish oil supplementation suppressed the production of pulmonary protective PGE2 in the bronchoalveolar lavage (BAL) while level of urinary metabolite of the PGE2 was increased. Our data suggest that augmented lung inflammation after fish oil supplementation may be due to the reduction of PGE2 production in the lung and these dichotomous results bring into question the role of fish oil supplementation in the treatment of asthma.
doi:10.1016/j.freeradbiomed.2009.05.033
PMCID: PMC2716436  PMID: 19501157
fish oil; ω-3 polyunsaturated fatty acids; asthma; free radicals; lung inflammation; mass spectrometry; Isoprostanes; prostaglandins; ovalbumin (OVA)
13.  Clinical Factors Associated with Plasma F2-Isoprostane Levels in HIV-Infected Adults 
HIV clinical trials  2009;10(3):181-192.
Purpose
Oxidant stress may be an effect of antiretroviral therapy (ART) or chronic HIV infection. Plasma F2-isoprostanes (F2-IsoP) reflect lipid peroxidation and oxidant stress and have been described in ART-associated toxicities. We explored factors associated with F2-IsoP in HIV-infected adults.
Methods
HIV-infected adults enrolled in this cross-sectional study were (a) on ART including zidovudine or stavudine but not non-nucleoside reverse transcriptase inhibitors (NNRTI), (b) on ART including NNRTI, or (c) not on ART. Plasma F2-IsoP levels were quantified by GC/MS, and clinical and laboratory data were collected at enrollment.
Results
Among 285 participants, 24% were female, 37% were African American, and 194 (68%) were on ART; 44 (23%) of whom were receiving efavirenz, 45 (23%) nevirapine, and 85 (44%) protease inhibitors. Median F2-IsoP was lower in those on NNRTI than those on ART without NNRTI (p = .02). In a multivariable model, factors independently associated with increased F2-IsoP were female sex (p =.002), higher BMI (p = .01), and heavy smoking (p =.004). There was a trend toward lower F2-IsoP among nevirapine users (p = .054).
Conclusions
Among HIV-infected adults, oxidant stress status differs by sex, BMI, smoking status, and perhaps specific ART. Prospective studies should better define relationships between oxidant stress and complications of HIV infection and its therapy.
doi:10.1310/hct1003-181
PMCID: PMC2891063  PMID: 19632957
antiretroviral therapy; F2-isoprostanes; highly active; HIV; lipid peroxidation; oxidative stress
14.  Cyclopentenone isoprostanes are novel bioactive products of lipid oxidation which enhance neurodegeneration 
Journal of neurochemistry  2006;97(5):1301-1313.
Oxidative stress and subsequent lipid peroxidation are involved in the pathogenesis of numerous neurodegenerative conditions, including stroke. Cyclopentenone isoprostanes (IsoPs) are novel electrophilic lipid peroxidation products formed under conditions of oxidative stress via the isoprostane pathway. These cyclopentenone IsoPs are isomeric to highly bioactive cyclopentenone prostaglandins, yet it has not been determined if these products are biologically active or are formed in the brain. Here we demonstrate that the major cyclopentenone IsoP isomer 15-A2t-IsoP potently induces apoptosis in neuronal cultures at submicromolar concentrations. We present a model in which 15-A2t-IsoP induced neuronal apoptosis involves initial depletion of glutathione and enhanced production of reactive oxygen species, followed by 12-lipoxygenase activation and phosphorylation of extracellular signal-regulated kinase 1/2 and the redox sensitive adaptor protein p66shc, which results in caspase-3 cleavage. 15-A2t-IsoP application also dramatically potentiates oxidative glutamate toxicity at concentrations as low as 100 nM, demonstrating the functional importance of these molecules in neurodegeneration. Finally, we employ novel mass spectrometric methods to show that cyclopentenone IsoPs are formed abundantly in brain tissue under conditions of oxidative stress. Together these findings suggest that cyclopentenone IsoPs may contribute to neuronal death caused by oxidative insults, and that their activity should perhaps be addressed when designing neuroprotective therapies.
doi:10.1111/j.1471-4159.2006.03797.x
PMCID: PMC2881557  PMID: 16638022
apoptosis; isoprostane; lipid peroxidation; 12-lipoxygenase; oxidative stress; p66shc
15.  Electrophilic Cyclopentenone Isoprostanes in Neurodegeneration 
Although oxidative stress has been implicated in the pathogenesis of numerous neurodegenerative conditions, the precise mechanisms by which reactive oxygen species (ROS) induce neuronal death are still being explored. The generation of reactive lipid peroxidation products is thought to contribute to ROS neurotoxicity. Isoprostanes (IsoPs), prostaglandin-like molecules formed in vivo via the ROS-mediated oxidation of arachidonic acid, have been previously demonstrated to be formed in increased amounts in the brains of patients with various neurodegenerative diseases. Recently, we have identified a new class of IsoPs, known as A2- and J2-IsoPs or cyclopentenone IsoPs, which are highly reactive electrophiles and form adducts with thiol-containing molecules, including cysteine residues in proteins and glutathione. Cyclopentenone IsoPs are favored products of the IsoP pathway in the brain and are formed abundantly after oxidant injury. These compounds also potently induce neuronal apoptosis by a mechanism which involves glutathione depletion, ROS generation, and activation of several redox-sensitive pathways that overlap with those involved in other forms of oxidative neurodegeneration. Cyclopentenone IsoPs also enhance neurodegeneration caused by other insults at biologically relevant concentrations. These data are reviewed, whereas new data demonstrating the neurotoxicity of J-ring IsoPs and a discussion of the possible role of cyclopentenone IsoPs as contributors to neurodegeneration are presented.
PMCID: PMC2881560  PMID: 17901550
Isoprostane; Oxidative stress; Lipid peroxidation; Neurodegeneration; Alzheimer’s disease; Arachidonic acid; Cyclopentenone; 12-lipoxygenase; Glutamate
16.  Low Sodium DASH Reduces Oxidative Stress and Improves Vascular Function in Salt-Sensitive Humans 
Journal of human hypertension  2009;23(12):826-835.
Salt induces oxidative stress in salt-sensitive (SS) animals and man. It is unknown in SS subjects if the low-sodium Dietary Approaches to Stop Hypertension (LS-DASH) reduces oxidative stress more than DASH, which is high in anti-oxidants. To assess the effects of DASH and LS-DASH on oxidative stress, 19 volunteers were studied after 3-weeks of a standardized usual low fruits and vegetables diet (ULFV), followed by 3-weeks on DASH (both diets ∼120 mmol Na+/day), then 3-weeks on low-sodium (LS)-DASH (60 mmol Na+/d). SS was defined as systolic blood pressure ≥5 mmHg lower on LS-DASH than DASH. In SS subjects (N=9), systolic blood pressure was lower on LS-DASH (111.0±2.0 mmHg) than DASH (118.0±2.2, p<0.01) and ULFV (122.3±2.7, p=0.002). In salt-resistant (SR) volunteers (N=10), systolic blood pressure was lower on DASH (113.0±1.6) than ULFV (119.0±1.8, p<0.05) but not LS-DASH (115.7±1.8). Urine F2-isoprostanes, a marker of oxidative stress, were lower in SS subjects on LS-DASH (1.69±0.24) than ULFV (3.09±0.50, p<0.05) and marginally lower than DASH (2.46±0.44, p<0.20). F2-isoprostanes were not different among the three diets in SR volunteers (2.18±0.29, 2.06±0.29, 2.27±0.53, respectively). Aortic augmentation index, a measure of vascular stiffness, was lower in SS subjects on LS-DASH than either DASH or ULFV, and lower on DASH than ULFV in SR volunteers. In SS but not SR subjects, LS-DASH is associated with lower values for F2-isorprostane and the aortic augmentation index. The results suggest that LS-DASH decreases oxidative stress, improves vascular function and lowers blood pressure in SS but not SR volunteers.
doi:10.1038/jhh.2009.32
PMCID: PMC2783838  PMID: 19404315
Blood pressure; Dietary Approaches to Stop Hypertension (DASH); salt sensitive; oxidative stress; vascular function
17.  Levels of Prostaglandin E Metabolite and Leukotriene E4 Are Increased in the Urine of Smokers. Evidence that Celecoxib Shunts Arachidonic Acid into the 5-Lipoxygenase Pathway 
Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) play a role in inflammation and carcinogenesis. Biomarkers that reflect tobacco smoke-induced tissue injury are needed. In this study, levels of urinary prostaglandin E metabolite (PGE-M) and leukotriene E4 (LTE4), biomarkers of the COX and 5-LO pathways, were compared in never smokers, former smokers and current smokers. The effects of celecoxib, a selective COX-2 inhibitor, on levels of PGE-M and LTE4 were determined. Baseline levels of PGE-M and LTE4 were positively associated with smoking status; levels of PGE-M and LTE4 were higher in current vs. never smokers. Treatment with celecoxib 200 mg bid for 6 ± 1 days led to a reduction in urinary PGE-M levels in all groups, but exhibited the greatest effect among subjects with high baseline PGE-M levels. Thus, high baseline PGE-M levels in smokers reflected increased COX-2 activity. In individuals with high baseline PGE-M levels, treatment with celecoxib led to a significant increase in levels of urinary LTE4, an effect that was not found in individuals with low baseline PGE-M levels. In conclusion, increased levels of urinary PGE-M and LTE4 were found in human smokers, a result that may reflect subclinical lung inflammation. In individuals with high baseline levels of PGE-M (elevated COX-2 activity), celecoxib administration shunted arachidonic acid into the pro-inflammatory 5-LO pathway. Because 5-LO activity and LTE4 have been suggested to play a role in cardiovascular disease, these results may help to explain the link between use of COX-2 inhibitors and cardiovascular complications.
doi:10.1158/1940-6207.CAPR-09-0005
PMCID: PMC2745265  PMID: 19336727
Smoking; Biomarker; Celecoxib; 5-lipoxygenase; cardiovascular toxicity
18.  20-Hydroxylation is the CYP-dependent and retinoid-inducible leukotriene B4 inactivation pathway in human and mouse skin cells 
Metabolic inactivation of leukotriene B4 (LTB4) is an innate mechanism to resolve tissue inflammation. We studied the nine Cyp4f genes in the mouse genome, measuring cutaneous transcript levels by real-time polymerase chain reaction, and LTB4 metabolism in mouse and human skin. Transcripts arising from Cyp4f13 and 4f16 ranked most abundant, Cyp4f14, 4f17, and 4f37 ranked least abundant, and Cyp4f18 and 4f39 ranked intermediate. Those from Cyp4f15 and Cyp4f40 were highly variable or too low to measure in some animals. Retinoic acid exposure induced microsomal LTB4 hydroxylation activities in mouse and human skin cells. Two NADPH-dependent LTB4 metabolites eluted identically with 20-OH and 20-COOH LTB4 reference standards. Collision induced dissociation of the precursor ion m/z 351 confirmed that LTB4 products from CYP4F3A and human epidermal keratinocytes are identical structurally to 20-OH LTB4. We conclude 20-hydroxylation is the major CYP-dependent LTB4 inactivation pathway in skin; this retinoid-inducible metabolic pathway has capacity to modulate tissue levels of pro-inflammatory lipids.
doi:10.1016/j.abb.2009.01.012
PMCID: PMC2687325  PMID: 19467632
19.  Oxidative Stress in Fibromyalgia and its Relationship to Symptoms 
Clinical rheumatology  2008;28(4):435-438.
Objective
Oxidative stress is thought to play a role in the pathogenesis of fibromyalgia. We examined the hypothesis that oxidative stress was increased in patients with fibromyalgia and related to the severity of symptoms.
Methods
Urinary F2 isoprostane excretion was measured in 48 patients with fibromyalgia and compared to those of 96 control subjects. In patients, we examined the association between oxidative stress and symptoms.
Results
Patients with fibromyalgia were significantly more symptomatic than control subjects, but urinary F2 isoprostane excretion did not differ significantly (2.3±1.9 vs 2.8±2.2 ng/mg creatinine) (p=0.16). In patients with fibromyalgia, F2-isoprostane excretion was associated with fatigue VAS (rho=0.30, p=0.04) but not with pain, quality of life, functional capacity, depression, number of tender points or overall impact of fibromyalgia.
Conclusion
oxidative stress is not increased in patients with fibromyalgia, but as was previously found in patients with systemic lupus erythematosus (SLE), oxidative stress was associated with fatigue.
doi:10.1007/s10067-008-1072-0
PMCID: PMC2654204  PMID: 19089486
fibromyalgia; oxidative stress; fatigue; F2 isoprostanes
20.  Drugs to Treat Systemic Lupus Erythematosus: Relationship between Current Use and Cardiovascular Risk Factors 
Archives of Drug Information  2008;1(1):23-28.
Objectives
Cardiovascular risk is increased in patients with systemic lupus erythematosus (SLE). Drugs used to treat SLE can modify traditional cardiovascular risk factors. We examined the effect of selected drugs used in the treatment of SLE on cardiovascular risk factors.
Methods
We compared systolic and diastolic blood pressure, serum lipid concentrations, glucose, homocysteine, and urinary F2-isoprostane concentrations in 99 patients with lupus who were either current users or non-users of systemic corticosteroids, antimalarials, non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 selective NSAIDs, azathioprine, and methotrexate. Multivariable adjustment was done with linear regression modeling using sex, age and disease activity (SLEDAI) as controlling variables.
Results
Serum triglyceride concentrations were higher (135.1 ± 61.4 vs. 95.3 ± 47.5 mg/dL, adjusted P = 0.003) in patients receiving corticosteroids. Homocysteine concentrations were marginally higher in patients receiving methotrexate (adjusted P = 0.08). Current use of either NSAIDs or COX-2 inhibitors was not associated with increased cardiovascular risk factors. Current hydroxychloroquine use was not associated with significant alterations in lipid profiles.
Conclusions
In a non-random sample of patients with SLE, current corticosteroid use was associated with increased triglyceride concentrations, but other drugs had little effect on traditional cardiovascular risk factors.
doi:10.1111/j.1753-5174.2007.00004.x
PMCID: PMC2817442  PMID: 20157365
Prednisone; Hydroxychloroquine; NSAID; COX-2 Inhibitor; Azathioprine, Methotrexate
21.  Oxidant Stress in HIV-Infected Women from the Women’s Interagency HIV Study 
Antiviral therapy  2009;14(6):763-769.
Background
Oxidant stress contributes to the pathogenesis of multiple conditions and can be assessed by measuring plasma F2-isoprostane concentrations. We hypothesized that oxidant stress is associated with plasma homocysteine concentration and risk factors for atherosclerosis in HIV-infected women.
Methods
We measured plasma F2-isoprostane concentrations in a cross-sectional study of 249 HIV-infected women attending the Bronx site of the Women’s Interagency HIV Study and assessed associations with plasma homocysteine concentration and other metabolic parameters by linear regression.
Results
In multivariate analysis, HCV viremia, waist circumference, homocysteine concentration, and serum aspartate transanimase level were positively associated with log F2-isoprostane concentration (all P < 0.005). There was a trend for an inverse association between log F2-isoprostane and CD4% (P = 0.06). Among women with HCV infection, the FIB-4 index, an indirect marker of liver fibrosis derived from routine laboratory tests, was positively associated with log F2-isoprostane concentration.
Conclusion
In this cross-sectional study of HIV-infected women, plasma F2-isoprostane concentration was positively associated with homocysteine concentration, as well as HCV infection, abdominal obesity, and aspartate transaminase level.
doi:10.3851/1290
PMCID: PMC2760028  PMID: 19812438
Oxidant stress; oxidative stress; F2-isoprostanes; homocysteine; HIV; HCV
22.  MARKERS OF OXIDATIVE STRESS AND SYSTEMIC VASOCONSTRICTION IN PREGNANT WOMEN DRINKING ≥ 48 GRAMS OF ALCOHOL PER DAY 
Background
The precise pathway by which alcohol causes the characteristic features of fetal alcohol spectrum disorders (FASD) is unknown. Proposed mechanisms for fetal injury from maternal alcohol use include cellular damage from oxidative stress and impaired fetal oxygenation related to maternal systemic vasoconstriction. Our objective was to compare levels of urinary markers of oxidative stress and systemic vasoconstriction between women consuming large amounts of alcohol during pregnancy and women who did not drink alcohol during pregnancy.
Methods
Pregnant women consuming ≥ 48g alcohol/day (n=29) on average and pregnant women who abstained from alcohol use (n=39) were identified using detailed interviews and home visits. Random maternal urine specimens were collected. Urinary levels of the oxidative stress marker, 8-isoprostane F2α, and of the vasoactive prostaglandin metabolites, 2,3-dinor-6-keto-prostaglandin F1α (a vasodilator) and 11-dehydro-thromboxane B2 (a vasoconstrictor), were measured using mass spectrometric methods. All analyte levels were corrected for urinary creatinine.
Results
In crude analyses, there was no significant difference in 8-isoprostane F2α between pregnant drinkers and nondrinkers (2.16 vs. 2.08 ng/mg creatinine respectively, P=.87). There were no significant differences between the drinking and non-drinking groups in levels of 2,3-dinor-6-keto-prostaglandin F1α (1.03 vs. 1.17 ng/mg creatinine repectively, P=.50), 11-dehydro-thromboxane B2 (0.72 vs. 0.59 ng/mg creatinine respectively, P=.21), or the ratio of vasodilatory metabolite to vasoconstrictive metabolite (1.73 vs. 2.72 respectively, P=.14). Adjusting for maternal age, marital status, smoking, and gestational age at sampling did not substantially alter the results.
Conclusion
Our results show no difference in levels of urinary eicosanoid markers of oxidative stress and systemic vasoconstriction between pregnant women who drink heavily and pregnant women who abstain. These findings speak against a role for maternal oxidative stress or systemic vasoconstriction in the pathogenesis of alcohol damage to the fetus.
doi:10.1111/j.1530-0277.2008.00773.x
PMCID: PMC2615682  PMID: 18715278
Alcohol; Pregnancy; Isoprostanes; Prostacyclin; Thromboxane; Fetal Alcohol Spectrum Disorders
23.  Effect of vitamins C and E on biomarkers of oxidative stress in nonsmokers 
Free radical biology & medicine  2008;45(4):377-384.
Background
Oxidative stress is elevated in obesity, and may be a major mechanism for obesity-related diseases.
Methods
Nonsmokers (n=396) were randomized to 1000 mg/day vitamin C, 800 IU/day vitamin E, or placebo, for two months. Treatment effect was examined in multiple regression analyses using an intention-to-treat approach.
Results
Vitamin C (p=0.001) and vitamin E (p=0.043) reduced plasma F2-isoprostanes. In the overall sample, changes from baseline were +6.8%, −10.6%, and −3.9% for placebo, vitamin C, and vitamin E groups, respectively. However, a significant interaction with baseline F2-isoprostane was found. When baseline F2-isoprostane was > 50 μg/mL, vitamin C reduced F2-isoprostane by 22% (p=0.01). Vitamin E reduced it by 9.8% (p=0.46). Below that cut-point, neither treatment produced further reductions. F2-isoprostane > 50 μg/mL was strongly associated with obesity, and was present in 42% of the sample. Change in malondialdehyde concentration was minimal.
Discussion
These findings suggest a role for vitamin C in reducing lipid peroxidation. Future research on effects of vitamins C or E on plasma F2-isoprostane should limit participants to those with baseline levels > 50 μg/mL. Further studies are needed to establish whether treatment with vitamins C or E in persons with concentrations above that cut-point could slow the development of cardiovascular disease.
doi:10.1016/j.freeradbiomed.2008.04.005
PMCID: PMC2750000  PMID: 18455517
Antioxidants; biomarkers; F2-isoprostane; obesity; oxidative stress; vitamin C; vitamin E; oxidative stress
25.  Identification of intact oxidation products of glycerophospholipids in vitro and in vivo using negative ion electrospray iontrap mass spectrometry 
Free radical-induced oxidation products of polyunsaturated fatty acids esterified to phospholipids have been implicated in a number of human diseases including atherosclerosis and neurodegenerative diseases. Some of these phospholipid oxidation products have potent biological activities and likely contribute to human pathophysiological conditions. Oxidation products have also been used as markers of oxidative stress in vivo. Identification and quantification of phospholipid oxidation products are often performed by analyzing the oxidized free fatty acid moieties after hydrolysis from the phospholipids head groups by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS). We now describe the definitive identification of intact oxidized products of glycerophospholipids including glycerophosphatidylcholine (GPC), glycerophosphatidylethanolamine (GPE), and glycerophosphatidylserine (GPS) in vitro and in vivo using iontrap MS. For these analyses, the negative ions of the oxidation products of phospholipids are fragmented to MSn and unequivocal structural characterization is obtained based on collision-induced dissociation (CID) of the sn-2 carboxylate ion. This technique overcomes the need to hydrolyze fatty acids from phospholipids in the analysis. The method has been used to identify a number of oxidation products of glycerophospholipids including hydroxyeicosatetraenoates (HETEs) and isoprostanes (IsoPs) esterified to different classes of glycerophospholipids in vitro and in vivo. These studies thus provide a new approach to identify the intact oxidation products of glycerolphospholipids.
doi:10.1002/jms.1542
PMCID: PMC2715274  PMID: 19125398
phospholipids; free radicals; oxidative stress; liquid chromatography–mass spectrometry; isoprostanes; iontrap MS

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