[Show abstract][Hide abstract] ABSTRACT: Chronic inflammation is a hallmark of HIV infection. Eicosanoids reflect inflammation, oxidant stress, and vascular health and vary by sex and metabolic parameters. Raltegravir (RAL) is an HIV-1 integrase inhibitor that may have limited metabolic effects. We assessed urinary F2-isoprostanes (F2-IsoPs), prostaglandin E2 (PGE-M), prostacyclin (PGI-M), and thromboxane B2 (TxB2) in HIV-infected women switching to RAL-containing antiretroviral therapy (ART). Thirty-seven women (RAL = 17; PI/NNRTI = 20) with a median age of 43 years and BMI 32 kg/m(2) completed week 24. TxB2 increased in the RAL versus PI/NNRTI arm (+0.09 versus -0.02; P = 0.06). Baseline PGI-M was lower in the RAL arm (P = 0.005); no other between-arm cross-sectional differences were observed. In the PI/NNRTI arm, 24-week visceral adipose tissue change correlated with PGI-M (rho = 0.45; P = 0.04) and TxB2 (rho = 0.44; P = 0.005) changes, with a trend seen for PGE-M (rho = 0.41; P = 0.07). In an adjusted model, age ≥ 50 years (N = 8) was associated with increased PGE-M (P = 0.04). In this randomized trial, a switch to RAL did not significantly affect urinary eicosanoids over 24 weeks. In women continuing PI/NNRTI, increased visceral adipose tissue correlated with increased PGI-M and PGE-M. Older age (≥50) was associated with increased PGE-M. Relationships between aging, adiposity, ART, and eicosanoids during HIV-infection require further study.
Mediators of Inflammation 06/2014; 2014:803095. DOI:10.1155/2014/803095 · 3.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: F(2)-Isoprostanes (IsoPs) are isomers of prostaglandin F(2α) formed from the nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Since discovery of these molecules by Morrow and Roberts in 1990, F(2)-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 F(2)-IsoPs in settings of increased oxygen tension. The protocol presented herein is the current methodology that our laboratory uses to quantify F(2)-IsoPs and IsoFs in biological tissues and fluids using gas chromatography/mass spectrometry (GC/MS). A variety of analytical procedures to measure F(2)-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 F(2)-IsoPs and IsoFs from a variety of biological sources including urine, plasma, tissues, cerebral spinal fluid, exhaled breath condensate, and amniotic fluid, among others.
Free Radical Biology and Medicine 10/2012; 59. DOI:10.1016/j.freeradbiomed.2012.09.030 · 5.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Obstetrics and Gynecology 07/2011; 118(1):121-34. DOI:10.1097/AOG.0b013e3182204eaa · 4.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cigarette smoking and bacterial infections are two major risk factors associated with preterm prelabor rupture of membranes (pPROM). We hypothesized that exposure of fetal membranes to cigarette smoke extracts might induce oxidative stress (OS) and fetal membrane apoptosis, culminating in an alternate pathway to that commonly activated by infection. To test this, we characterized the production of prostanoids and biomarkers of apoptosis in normal term human fetal membrane explant cultures. Fetal membrane explants collected at term (from cesarean deliveries, not in labor) were stimulated with cigarette smoke extract (CSE) for 24 h. Two classes of prostanoids, F2-Isoprostane (F2-IsoP), a marker of OS and PGF2α, a classical uterotonin, were measured by gas chromatography/mass spectrometry. Western blot analyses of tissue lysates were performed to quantify the anti-apoptotic protein Bcl2 and actin (as a control). Fetal membrane apoptosis was detected by immunohistochemistry for active caspase 3 and confirmed by TUNEL staining for nuclear fragmentation. CSE exposure resulted in significantly more F2-IsoP production from fetal membranes (242.8 ± 79.3 pg/ml/mg of total membrane protein) compared to unstimulated controls (131.5 ± 53.1 pg/ml/mg; p < 0.0001). By contrast, PGF2α was not different in CSE vs. controls (1083 ± 527 vs. 1136 ± 835 pg/ml/mg of protein; p = 0.80). CSE-exposed tissues demonstrated a dose-dependent decrease in Bcl2 expression and increases in active caspase 3 and nuclear fragmentation in both amnion and chorion cells compared to controls. In summary, fetal membranes exposed to CSE manifest evidence of OS and apoptosis. The differential pattern of prostanoid production observed in this study supports the hypothesis that an alternate non-inflammatory pathway mediated by OS and apoptosis in pPROM may promote proteolysis resulting in membrane weakening and rupture.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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).
Our study results suggest that these four urinary biomarkers have relatively low intra-person variation over a 1-year period.
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.
[Show abstract][Hide abstract] ABSTRACT: Oxidant stress contributes to the pathogenesis of multiple conditions and can be assessed by measuring plasma F(2)-isoprostane concentrations. We hypothesized that oxidant stress is associated with plasma homocysteine concentration and risk factors for atherosclerosis in HIV-infected women.
We measured plasma F(2)-isoprostane concentrations in a cross-sectional study of 249 HIV-infected women attending the Bronx (NY, USA) site of the Women's Interagency HIV Study and assessed associations with plasma homocysteine concentration and other metabolic parameters by linear regression.
In multivariate analysis, hepatitis C virus (HCV) viraemia, waist circumference, homocysteine concentration and serum aspartate aminotransferase level were positively associated with log F(2)-isoprostane concentration (all P<0.005). There was a trend for an inverse association between log F(2)-isoprostane and CD4(+) T-cell percentage (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 F(2)-isoprostane concentration.
In this cross-sectional study of HIV-infected women, plasma F(2)-isoprostane concentration was positively associated with homocysteine concentration, as well as HCV infection, abdominal obesity and aspartate aminotransferase level.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Oxidant stress has been implicated in a wide variety of disease processes. One method to quantify oxidative injury is to measure lipid peroxidation. Quantification of a group of prostaglandin F(2alpha)-like compounds derived from the nonezymatic oxidation of arachidonic acid, termed the F2-isoprostanes (F2-IsoPs), provides an accurate assessment of oxidative stress both in vitro and in vivo. In fact, in a recent independent study sponsored by the National Institutes of Health (NIH), F2-IsoPs were shown to be the most reliable index of in vivo oxidant stress when compared against other well known biomarkers. This protocol details our laboratory's method to quantify F2-IsoPs in biological fluids and tissues using gas chromatography-mass spectrometry (GC-MS). This procedure can be completed for 12-15 samples in 6-8 h.
[Show abstract][Hide abstract] ABSTRACT: Oxidant stress has been implicated in a wide variety of disease processes. One method to quantify oxidative injury is to measure lipid peroxidation. Quantification of a group of prostaglandin F(2)-like compounds derived from the nonezymatic oxidation of arachidonic acid, termed the F(2)-isoprostanes (F(2)-IsoPs), provides an accurate assessment of oxidative stress both in vitro and in vivo. In fact, in a recent National Institutes of Health-sponsored independent study, F(2)-IsoPs were shown to be the most reliable index of in vivo oxidant stress when compared against other well-known biomarkers. This article summarizes current methodology used to quantify these molecules. Our laboratory's method to measure F(2)-IsoPs in biological fluids and tissues using gas chromatography-mass spectrometry is detailed herein. In addition, other mass spectrometric approaches, as well as immunological methods to measure these compounds, are discussed. Finally, the utility of these molecules as in vivo biomarkers of oxidative stress is summarized.
Methods in Enzymology 02/2007; 433:113-26. DOI:10.1016/S0076-6879(07)33006-1 · 2.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antiplatelet therapies improve endothelial function in atherosclerosis, suggesting that platelets regulate vascular nitric oxide (NO) bioactivity in vivo. Herein, washed platelets consumed NO on activation in an aspirin-sensitive manner, and aspirin enhanced platelet NO responses in vitro. To examine whether in vivo aspirin can inhibit platelet NO consumption, a double-blind placebo-controlled study was conducted. After a 2-week nonsteroidal anti-inflammatory drug (NSAID)-free period, healthy men were randomly assigned and administered aspirin (75 mg/d orally) or identical placebo for 14 days, then crossed over to the opposite arm. Following in vivo aspirin, NO consumption by platelets was inhibited 91%. Rate of onset and recovery following aspirin withdrawal was consistent with cyclooxygenase 1 (COX-1) inhibition. In a small substudy, NO consumption by platelets from postmenopausal women was faster in hypercholesterolemics and less sensitive to aspirin (ie, 39% versus 76% inhibition for hypercholesterolemics or normocholesterolemics, respectively). However, 150 mg aspirin/day increased inhibition of NO consumption by platelets of hypercholesterolemics to 80%. Comparisons of platelet COX-1 or -2 expression and urinary 11-dehydro-thromboxane B2 excretion suggested that aspirin was less able to block platelet activation in vivo in hypercholesterolemia. In conclusion, aspirin inhibits NO consumption by platelets from healthy subjects, but its beneficial effects on NO bioactivity may be compromised in some hypercholesterolemic patients.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Quantitative biomarkers of oxidative damage, such as the F(2)-isoprostanes (IsoPs) and F(4)-neuroprostanes (F(4)-NeuroPs), may be useful in assessing progression and response to therapeutics in patients with Alzheimer's disease. F(2)-IsoPs and F(4)-NeuroPs are reproducibly increased in brain and cerebrospinal fluid of Alzheimer's disease patients; however, results in blood and urine have been conflicting. We tested the hypothesis that F(2)-IsoPs and F(4)-NeuroPs in plasma or urine quantitatively reflect oxidative damage to the central nervous system. Our results showed that urine levels of F(2)-IsoPs or their major metabolite were not significantly different between 56 Alzheimer's disease patients and 34 controls. In addition, urine and cerebrospinal fluid F(2)-IsoP levels in 32 Alzheimer's disease patients did not correlate. Supporting these conclusions, elevated rat cerebral F(2)-IsoPs and F(4)-NeuroPs after systemic exposure to kainic acid were not associated with a significant change in their plasma or urine levels. These results show that plasma and urine F(2)-IsoPs and F(4)-NeuroPs do not accurately reflect central nervous system levels of these biomarkers and are not reproducibly elevated in body fluids outside of central nervous system in Alzheimer's disease patients. These results should guide the organization of clinical trials now being planned for patients with Alzheimer's disease.
Annals of Neurology 08/2002; 52(2):175-9. DOI:10.1002/ana.10272 · 11.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Numerous post mortem studies have demonstrated increased accumulation of lipid peroxidation products in diseased regions of Alzheimer's disease (AD) brain; however, few have used techniques that quantify the magnitude of lipid peroxidation in vivo. F(2)-isoprostanes (F(2)-IsoP's) are exclusive products of free radical-mediated peroxidation of arachidonic acid, and their quantification has been widely used as an in vivo biomarker of the magnitude of lipid peroxidation. We have determined F(2)-IsoP concentrations in lateral ventricular fluid (VF) from 23 AD and 12 age-matched controls and correlated these with neuropathological and genetic markers of AD. VF F(2)-IsoP levels were significantly elevated in AD patients compared with controls (p < 0.01) and were significantly correlated with three different measures of brain degeneration: reduction in brain weight (p < 0.01), degree of cortical atrophy (p < 0.01), and Braak stage (p = 0.02). When analysis was restricted to AD patients only, VF F(2)-IsoP levels still were significantly correlated to reduction in brain weight and degree of cortical atrophy (p < 0.05). VF F(2)-IsoP concentrations were not related to density of neuritic plaques or neurofibrillary tangles in seven brain regions, or to the number of epsilon4 alleles of the apolipoprotein E gene (APOE). These data suggest that the magnitude of brain lipid peroxidation is closely related to the extent of brain degeneration in AD but is not significantly influenced by the density of neuritic plaques or neurofibrillary tangles, or the number of epsilon4 alleles of APOE.
American Journal Of Pathology 10/1999; 155(3):863-8. DOI:10.1016/S0002-9440(10)65185-1 · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Increased expression of cyclooxygenase (COX) and overproduction of prostaglandins (PGs) have been implicated in the development and progression of colorectal cancer (CRC). Nonsteroidal anti-inflammatory agents (NSAIDS) inhibit growth of various CRC cell lines by both COX-dependent and COX-independent pathways. To specifically examine the effect of COX and PGs on proliferation in CRC cells, we introduced an antisense COX-2 cDNA construct under the control of a tetracycline (Tc)-inducible promoter into a CRC cell line, HCA-7, Colony 29 (HCA-7) that expresses COX and produces PGs. In the presence of Tc, PG production in COX-depleted cells was reduced 99.8% compared with either uninduced transfectants or parental HCA-7 cells. This decrease in PG production was associated with a concomitant 60% reduction in DNA replication. Subsequently, we examined the effects of various PGs to modulate cell growth in COX-depleted HCA-7 or COX-null HCT-15 cells by quantifying [3H]thymidine incorporation and/or growth in collagen gels. We report that J-series cyclopentenone PGs, particularly PGJ2 and 15-deoxy-delta12,14-PGJ2, induce proliferation of these cells at nanomolar concentrations. Lipids extracted from parental HCA-7 cell conditioned medium stimulated mitogenesis in COX-depleted HCA-7 cells and COX-null HCT-15 cells. Using chromatographic and mass spectrometric approaches, we were able to detect PGJ2 in conditioned medium from parental HCA-7 cells. Taken together, these findings implicate a role for cyclopentenone PGs in CRC cell proliferation.
Cancer Research 07/1999; 59(11):2739-46. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The discovery of IsoPs as products of nonenzymatic lipid peroxidation has opened up new areas of investigation regarding the role of free radicals in human physiology and pathophysiology. The quantification of IsoPs as markers of oxidative stress status appears to be an important advance in our ability to explore the role of free radicals in the pathogenesis of human disease. An important need in the field of free-radical medicine is information regarding the clinical pharmacology of antioxidant agents. Because of the evidence implicating free radicals in the pathogenesis of a number of human diseases, large clinical trials are planned or underway to assess whether antioxidants can either prevent the development or ameliorate the pathology of certain human disorders. However, data regarding the most effective doses and combination of antioxidant agents to use in these clinical trials is lacking. As mentioned previously, administration of antioxidants suppresses the formation of IsoPs, even in normal individuals. Thus, measurement of IsoPs may provide a valuable approach to define the clinical pharmacology of antioxidants. In addition to being markers of oxidative stress, several IsoPs possess potent biological activity. The availability of additional IsoPs in synthetic form should broaden our knowledge concerning the role of these molecules as mediators of oxidant stress. Despite the fact that considerable information has been obtained since the initial report of the discovery of IsoPs , much remains to be understood about these molecules. With continued research in this area, we believe that much new information will emerge that will open up additional important new areas for future investigation.
Drug Metabolism Reviews 03/1999; 31(1):117-39. DOI:10.1081/DMR-100101910 · 6.29 Impact Factor