Stephanie C. Sanchez

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 F2α-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 laboratorys 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.

Peripheral F2‐isoprostanes and F4‐neuroprostanes are not increased in Alzheimer's disease

Quantitative biomarkers of oxidative damage, such as the F2‐isoprostanes (IsoPs) and F4‐neuroprostanes (F4‐NeuroPs), may be useful in assessing progression and response to therapeutics in patients with Alzheimers disease. F2‐IsoPs and F4‐NeuroPs are reproducibly increased in brain and cerebrospinal fluid of Alzheimers disease patients; however, results in blood and urine have been conflicting. We tested the hypothesis that F2‐IsoPs and F4‐NeuroPs in plasma or urine quantitatively reflect oxidative damage to the central nervous system. Our results showed that urine levels of F2‐IsoPs or their major metabolite were not significantly different between 56 Alzheimers disease patients and 34 controls. In addition, urine and cerebrospinal fluid F2‐IsoP levels in 32 Alzheimers disease patients did not correlate. Supporting these conclusions, elevated rat cerebral F2‐IsoPs and F4‐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 F2‐IsoPs and F4‐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 Alzheimers disease patients. These results should guide the organization of clinical trials now being planned for patients with Alzheimers disease.

Quantification of F2-isoprostanes in biological fluids and tissues as a measure of oxidant 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(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 laboratorys 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.

THE ISOPROSTANES: UNIQUE PROSTAGLANDIN-LIKE PRODUCTS OF FREE-RADICAL-INITIATED LIPID PEROXIDATION*

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 [6], 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.

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

Omega-3 (ω-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 ω-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 A2-like and prostaglandin J2-like compounds, termed A2/J2-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 A3/J3-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 A3/J3-IsoPs. We found that levels of these molecules increase ∼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.

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, PGF2&agr;, and PGD2 and for 6-keto-PGF1&agr; (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 PGF2&agr; was higher in preterm birth 6-keto-PGF1&agr; 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 PGF2&agr;. LEVEL OF EVIDENCE: III

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 F2-isoprostanes (F2-IsoPs), 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M), a major metabolite of F2-IsoPs, prostaglandin E2 metabolite (PGE-M), and leukotriene E4 (LTE4) 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 Mens 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 F2-IsoPs, 15-F2t-IsoP-M, PGE-M, and LTE4 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 F2-IsoPs, 15-F2t-IsoP-M, PGE-M, and LTE4. Except for high correlations between F2-IsoPs and 15-F2t-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 F2-IsoPs, 15-F2t-IsoP-M, PGE-M, and LTE4 could be useful as biomarkers of oxidative stress and inflammation status for epidemiologic studies. Cancer Epidemiol Biomarkers Prev; 19(4); 947–52. ©2010 AACR.

Formation of Novel Isoprostane-Like Compounds from Docosahexaenoic Acid

Isoprostanes (IsoPs) are prostaglandin (PG)-like compounds that are formed non-enzymatically in vivo by free radical-induced peroxidation of arachidonic acid.1 Formation of these compounds proceeds through bicyclic endoperoxide intermediates resembling PGH2. The endoperoxide intermediates are then reduced to PGF2-like compounds termed F2-IsoPS or undergo rearrangement to form D-ring and E-ring IsoPs (D2/E2-IsoPs)2 and thromboxane-like compounds (isothromboxanes).3 Unlike cyclooxygenase derived PGs in which arachidonic acid must first be released from phospholipids prior to oxygenation, IsoPs are formed completely in situ esterified in lipids and are subsequently released by a phospholipase(s) A2.4 Since the identification of these compounds, we have accumulated a large body of evidence showing that quantification of IsoPs provides an accurate measure of lipid peroxidation in vitro and in vivo.5,6 Further, at least three of these compounds possess biological activity1,2,7 and thus may mediate some of the adverse effects associated with oxidant stress.

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/m2 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.