Identification of novel F2-isoprostane metabolites by specific UDP-glucuronosyltransferases.

UDP-glucuronosyltransferases (UGTs) catalyze the conjugation of glucuronic acid with endogenous and exogenous lipophilic small molecules to facilitate their inactivation and excretion from the body. This represents approximately 35 % of all phase II metabolic transformations. Fatty acids and their oxidized eicosanoid derivatives can be metabolized by UGTs. F2-isoprostanes (F2-IsoPs) are eicosanoids formed from the free radical oxidation of arachidonic acid. These molecules are potent vasoconstrictors and are widely used as biomarkers of endogenous oxidative damage. An increasing body of evidence demonstrates the efficacy of measuring the ß-oxidation metabolites of F2-IsoPs rather than the unmetabolized F2-IsoPs to quantify oxidative damage in certain settings. Yet, the metabolism of F2-IsoPs is incompletely understood. This study sought to identify and characterize novel phase II metabolites of 15-F2t-IsoP and 5-epi-5-F2t-IsoP, two abundantly produced F2-IsoPs, in human liver microsomes (HLM). Utilizing liquid chromatography-mass spectrometry, we demonstrated that glucuronide conjugates are the major metabolites of these F2-IsoPs in HLM. Further, we showed that these molecules are metabolized by specific UGT isoforms. 15-F2t-IsoP is metabolized by UGT1A3, 1A9, and 2B7, while 5-epi-5-F2t-IsoP is metabolized by UGT1A7, 1A9, and 2B7. We identified, for the first time, the formation of intact glucuronide F2-IsoPs in human urine and showed that F2-IsoP glucuronidation is reduced in people supplemented with eicosapentaenoic and docosahexaenoic acids for 12 weeks. These studies demonstrate that endogenous F2-IsoP levels can be modified by factors other than redox mechanisms.

Urinary eicosanoid metabolites in HIV-infected women with central obesity switching to raltegravir: an analysis from the women, integrase, and fat accumulation trial.

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.

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.

Oxidant stress in HIV-infected women from the Women's Interagency HIV Study.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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 F2-isoprostanes as a biomarker of oxidative stress.

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.

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.