The biochemistry of the isoprostane, neuroprostane, and isofuran pathways of lipid peroxidation

F2-isoprostanes are prostaglandin F2-like compounds that are formed nonenzymatically by free radical mediated peroxidation of arachidonic acid. Intermediate in the pathway of the formation of isoprostanes are labile prostaglandin H2-like bicyclic endoperoxides (H2-isoprostanes), which are reduced to F2-isoprostanes and also undergo rearrangement in vivo to form E-ring and D-ring isoprostanes, isothromboxanes, and highly reactive acyclic gamma-ketoaldehdyes (isoketals). Docosahexaenoic acid (C22:6omega3) is highly enriched in neurons in the brain and is highly susceptible to oxidation. Free radical mediated oxidation of docosahexaenoic acid results in the formation of isoprostane-like compounds (neuroprostanes). F4- and E4/D4-neuroprostanes as well as neuroketals have been shown to be produced in vivo. Finally, we recently discovered a new pathway of lipid peroxidation that forms compounds with a substituted tetrahydrofuran ring (isofurans). Oxygen concentrations differentially modulate the formation of isoprostanes and isofurans; at elevated oxygen concentrations, the formation of isofurans is favored whereas the formation of isoprostanes is disfavored.     

Isoprostanes and related products of lipid peroxidation in neurodegenerative diseases

Lipid peroxidation is a major outcome of free radical-mediated injury to brain, where it directly damages membranes and generates a number of oxidized products. Some of the chemically and metabolically stable oxidation products are useful in vivo biomarkers of lipid peroxidation. These include the isoprostanes (IsoPs) and isofurans (IsoFs), derived from arachidonic acid (AA), and neuroprostanes (NeuroPs), derived from docosahexaenoic acid (DHA). We have shown increased levels of IsoPs, NeuroPs, and IsoFs in diseased regions of brain from patients who died from advanced Alzheimer's disease (AD) or Parkinson's disease (PD). Increased cerebrospinal fluid (CSF) levels of IsoPs are present in patients with AD or Huntington's disease (HD) early in the course of their illness, and CSF IsoPs may improve the laboratory diagnostic accuracy for AD. In contrast, quantification of IsoPs in plasma and urine of AD patients has yielded inconsistent results. These results indicate that brain lipid peroxidation is a potential therapeutic target early in the course of AD and HD, that CSF IsoPs may aid in the assessment of anti-oxidant experimental therapeutics and laboratory diagnosis of AD.     

Neurofurans, novel indices of oxidant stress derived from docosahexaenoic acid

Isoeicosanoids are free radical-catalyzed isomers of the enzymatic products of arachidonic acid. They are formed in situ in cell membranes, are cleaved, circulate, and are excreted in urine. Isomers of prostaglandin F(2alpha), the F(2)-isoprostanes, have emerged as sensitive indices of lipid peroxidation in vivo. Analogous compounds formed from docosahexaenoic acid (DHA) are termed neuroprostanes and are more abundant than isoprostanes (iPs) in brain. Isofurans are another class of isoeicosanoids characterized by a substituted tetrahydrofuran ring. They are preferentially formed, relative to iPs, under conditions of elevated oxygen tension. Here, we report the discovery of neurofurans (nFs), the analogous family of compounds formed from DHA. Formation of nFs is characterized by mass spectrometry and confirmed by oxidation of DHA in vitro and following CCl(4) administration in liver in vivo. It is demonstrated that the levels of nFs are elevated in the brain cortex of a mouse model of Alzheimer disease and are depressed in mouse brain cortex by deletion of p47(phox), an essential component of the phagocyte NADPH oxidase. Measurement of the nFs may ultimately prove useful in diagnosis, timing, and selection of dose in the treatment and chemoprevention of neurodegenerative disease.     

Urinary isoprostane excretion is not confounded by the lipid content of the diet.

This study aims to determine if isoprostanes accurately reflect in vivo lipid peroxidation or whether they are influenced by the lipid content of the diet. Isoprostanes were measured in urine of healthy subjects under different conditions of lipid intake and under conditions of oxidative stress (fasting). We found that isoprostanes were not influenced by the lipid content of the diet: the urinary level remained constant over 24 h as well as over 4 consecutive days when switching from high to low lipid intake. Urinary isoprostane excretion was increased by 40% following a 24 h fast. We concluded that urinary isoprostane excretion reflects endogenous lipid peroxidation in vivo.     

Isoprostanes: an overview and putative roles in pulmonary pathophysiology

Isoprostanes are produced during peroxidation of membrane lipids by free radicals and reactive oxygen species. Initially, they were recognized as being valuable markers of oxidative stress, and in the past 10 years, dozens of disease states and experimental conditions with diverse etiologies have been shown to be associated with marked increases in urinary, plasma, and tissue levels of isoprostanes. However, they are not just mere markers; they evoke important biological responses on virtually every cell type found within the lung, and these responses exhibit compound-, tissue-, and species-related variations. In fact, the isoprostanes may mediate many of the features of the disease states for which they are used as indicators. In this review, I describe the chemistry, metabolism, and pharmacology of isoprostanes, with a particular emphasis on pulmonary cell types, and the possible roles of isoprostanes in pulmonary pathophysiology.     

A nomenclature system for isofurans

Recently, the isolation of a new class of human arachidonic acid oxidation products, the isofurans, was reported. These are produced in vivo by a free radical mechanism independent of the cyclooxygenase enzymes. Because these compounds are tetrahydrofuran derivatives that are related biosynthetically to the isoprostanes, they were termed isofurans. There are eight different isofuran regioisomers, each of which can exist as 16 racemic diastereomers. Thus, 256 enantiomerically-pure isofurans can be formed. These molecules are of interest as measurement of isofurans provides a sensitive index of free-radical induced lipid peroxidation in vivo under conditions of elevated oxygen tension. They also, in analogy to isoprostanes, may have potent biological activity. To explore this, the chemical synthesis of the IsoFs has been initiated. As a result, there is a need for a systematic nomenclature for this class of natural products. A facile system that will allow the ready differentiation of each of the isomeric structures comprising the family of isofurans is presented.     

Isoprostanes in amniotic fluid: a predictive marker for fetal growth restriction in pregnancy

Isoprostanes are markers of free radical-catalyzed lipid peroxidation. Evidence suggests that oxidative stress occurs in pregnancies with fetal growth restriction (FGR). The aim of this study was to analyze F2-isoprostanes in amniotic fluid of FGR pregnancies. We tested the hypothesis that F2-isoprostanes are reliable markers to distinguish FGR pregnancies from normal ones and appropriate-for-gestational-age (AGA) from small-for-gestational-age (SGA) newborns. F2-isoprostanes levels were measured by colorimetric enzyme immunoassay in the amniotic fluid of 77 pregnancies with normal fetal growth (group I) and 37 with FGR (group II). Fetal biometry and Doppler measurements were obtained using an ATL HDI 3000 ultrasound system. Isoprostanes were higher in group II than group I. The ROC curve distinguished group I from group II, showing 100% sensitivity and 88.3% specificity at a cutoff of 94 pg/ml. There were no statistical differences in isoprostanes levels between AGA and SGA newborns in group II. The area under the ROC curve drawn to distinguish AGA and SGA newborns showed a sensitivity of 100% and a specificity of 72.3% at a cutoff of 94 pg/ml. The relative risk index indicated a 8.05 times higher risk of birth weight below the 3rd percentiles in group II than in group I. High isoprostanes concentrations can be detected in the amniotic fluid of FGR pregnancies and the assay of isoprostanes in amniotic fluid is a reliable assessment of fetal oxidative stress. Common use of this predictive marker in obstetrics will improve the ability of clinicians to identify those fetuses who will be born SGA or with a birth weight below the 25th percentile.     

Lipid peroxidation in aging brain and Alzheimer's disease

Lipid peroxidation is one of the major outcomes of free radical-mediated injury that directly damages membranes and generates a number of secondary products, both from fission and endocyclization of oxygenated fatty acids that possess neurotoxic activity. Numerous studies have demonstrated increased lipid peroxidation in brain of patients with Alzheimer's disease (AD) compared with age-matched controls. These data include quantification of fission and endocyclized products such as 4-hydroxy-2-nonenal, acrolein, isoprostanes, and neuroprostanes. Immunohistochemical and biochemical studies have localized the majority of lipid peroxidation products to neurons. A few studies have consistently demonstrated increased cerebrospinal fluid (CSF) levels of isoprostanes in AD patients early in the course of their dementia, and one study has suggested that CSF isoprostanes may improve the laboratory diagnostic accuracy for AD. Similar analyses of control individuals over a wide range of ages indicate that brain lipid peroxidation is not a significant feature of usual aging. Quantification of isoprostanes in plasma and urine of AD patients has yielded inconsistent results. These results indicate that brain lipid peroxidation is a potential therapeutic target in probable AD patients, and that CSF isoprostanes may aid in the assessment of antioxidant experimental therapeutics and the laboratory diagnosis of AD.     

Isoprostanes in fetal and neonatal health and disease

Isoprostanes are prostaglandin-like bioactive molecules generated via nonenzymatic peroxidation of lipid membrane-derived arachidonic acid by free radicals and reactive oxygen species. Their cognate receptors, biological actions, and signaling pathways are poorly understood. Aside from being sensitive and specific biomarkers of oxidative stress, E- and F-ring isoprostanes have important biological functions and likely mediate many of the disease-related pathological changes for which they are used as indicators. The biochemical pathways involved in isoprostane formation, their pathogenetic relevance to adult disease states, and their biological function are addressed. Developmentally, plasma and tissue content data show that isoprostane levels are highest during fetal and early neonatal life, when compared with adults. As such, the available data suggesting that isoprostanes play an important biological role, as well as possibly actively participate in the regulation of pulmonary vascular tone and the transition from fetal to postnatal life, are here reviewed. Lastly, the association between isoprostanes and certain neonatal clinical conditions is addressed. Although its existence has been recognized for almost 20 years, little is known about the critical importance of isoprostanes during fetal life and immediate neonatal period. This review is an attempt to bridge this knowledge gap.     

Expression of angiostatin and its related factors in the plasma of newborn pigs with hypoxia and reoxygenation

Little is known about angiostatin and its related factors in the hypoxia-reoxygenation of neonates. In this study we compared the effect of 21% and 100% reoxygenation on temporal changes in the plasma level of these factors in newborn piglets subjected to hypoxia. Newborn piglets were subjected to 2 h hypoxia followed by 1 h of reoxygenation with either 21% or 100% oxygen and observed for 4 days. On day 4 of recovery in 100% hypoxic-reoxygenated group, there were increases in total angiostatin, plasminogen/plasmin and MMP-2 levels, and decreases in VEGF levels (vs. respective baseline levels, all P <0.001), whereas no significant temporal changes were found in the 21% hypoxic-reoxygenated and sham-operated groups. Angiostatin levels correlated positively with the levels of MMP-2 and HIF-1alpha and negatively with VEGF levels in 100% hypoxic-reoxygenated group (all P <0.05). In comparison to 21% oxygen, neonatal resuscitation with 100% oxygen was found to increase the levels anti-angiogenic factors.     

Relationship of urinary isoprostanes to prostate cancer occurence

To estimate the oxidative stress in patients with prostate cancer and in a control group, we used the biomarker of lipid peroxidation?Cisoprostanes (8-isoPGF₂) and the level of selected antioxidants (glucose and uric acid [UA]). The level of urinary isoprostanes was determined in patients and controls using an immunoassay kit according to the manufacturer??s instruction. The levels of UA and glucose were also determined in serum by the use of UA Assay Kit and Glucose Assay Kit. We observed a statistically increased the level of isoprostanes in urine of patients with prostate cancer in compared with a control group. The concentration of tested antioxidants in blood from patients with prostate cancer was also higher than in healthy subjects. Moreover, our experiments indicate that the correlation between the increased amount of UA and the lipid peroxidation exists in prostate cancer patients (in all tested groups). Prostate cancer risk by urinary isoprostanes level was analyzed, and a positive association was found (relative risk for highest vs. lowest quartile of urinary isoprostanes?=?1.6; 95?% confidence interval 1.2?C2.4; p for trend?=?0.03). We suggest that reactive oxygen species induce peroxidation of unsaturated fatty acid in patients with prostate cancer, and the level of isoprostanes may be used as a non-invasive marker for determination of oxidative stress. We also propose that UA may enhance the oxidative stress in patients with prostate cancer.     

The pulmonary biology of isoprostanes

Isoprostanes are widely recognized as useful markers of membrane lipid peroxidation. It seems to be less well appreciated, however, that they also elicit important biological responses, even though this was first shown at the same time that they were introduced as markers of oxidative stress. The past several years have seen the list of cells/tissues which are sensitive to isoprostanes grow considerably: in fact, as we summarize here, there is now evidence that essentially every cell type in the lung responds in some pathologically relevant way to isoprostanes. In this sense, they might well be considered as not just markers of oxidative stress and inflammation, but also as a novel group of inflammatory mediators. Moreover, in addition to their pathological effects, we summarize here the evidence which has led us to hypothesize that isoprostanes could play an important role in vascular smooth muscle physiology as "endothelium-derived hyperpolarizing factors."     

LC-MS/MS methods for the detection of isoprostanes (iPF2alpha-III and 8,12-iso-iPF2alpha-VI) as biomarkers of CCl4-induced oxidative damage to hepatic tissue

Isoprostanes are formed after peroxidation of arachidonic acid and are promising biomarkers for reactive oxygen species. A LC-MS/MS based method was developed for the quantitation of two isoprostanes (iPF2alpha-III and 8,12-iso-iPF2alpha-VI) in hepatocytes, tissue and urine samples of rats. A column switching method was used to reduce sample preparation to a minimum. Precision was 9.4% and accuracy was between 96 and 114% for free iPF2alpha-III in tissue at concentrations from 1.9 to 6.1 ng/g. Treatment of rats with CCl4 to induce oxidative stress resulted in a dose-dependent increase (two- to three-fold) of iPF2alpha-III and 8,12-iso-iPF2alpha-VI in liver and kidney. For both isoprostanes an increase of four- to five-fold was observed in CCl4 treated hepatocytes and six- to eight-fold in CCl4 treated and glutathione depleted hepatocytes. In conclusion, the presented method is sensitive, specific and precise to be applied for the quantitation of iPF2alpha-III and 8,12-iso-iPF2alpha-VI which are shown to increase by CCl4 treatment in vitro and in vivo.     

Kinetics of biomarkers: biological and technical validity of isoprostanes in plasma

Summary. Isoprostanes, non-enzymatic peroxidation products of arachidonic acid, are attractive biomarkers of oxidative stress in research in biology, medicine and nutrition. For the appropriate use of biomarkers it is required that these are both biologically and technically valid. Whereas the biological validity of isoprostanes is well-established, it is technically quite complicated to measure isoprostanes and its metabolites in body fluids, and its rapid disappearance from plasma may hamper practical application. This paper shortly introduces isoprostanes as a biomarker for studies with humans, describes a novel fast and sensitive method for measuring isoprostanes in plasma by high-performance liquid chromatography and tandem mass spectrometry, and provides several examples of the use of the method in studies in humans. By taking care of the biological and technical validity of this biomarker it is possible to establish the antioxidant effects of some food ingredients in studies with human volunteers.     

Pulmonary hemodynamics and vascular reactivity in asphyxiated term lambs resuscitated with 21 and 100% oxygen

An increase in oxygen tension is an important factor in decreasing pulmonary vascular resistance (PVR) at birth. Birth asphyxia results in acidosis and increased PVR. We determined the effect of resuscitation with 21 vs. 100% O(2) on pulmonary hemodynamics, pulmonary arterial (PA) reactivity, and oxidant stress in a lamb model of in utero asphyxia. Term fetal lambs were acutely asphyxiated by intrauterine umbilical cord occlusion for 10 min resulting in acidosis (pH 6.96 ± 0.05 and Pco(2) 103 ± 5 Torr), bradycardia, systemic hypotension, and increased PVR. Lambs were treated with 30 min of resuscitation with 21% or 100% O(2) (n = 6 each). Pa(O(2)) was significantly elevated with 100% O(2) resuscitation compared with 21% O(2) (430 ± 38 vs. 64 ± 8 Torr), but changes in pH and Pa(CO(2)) were similar. The 100% O(2) induced greater increase in pulmonary blood flow and decrease in PVR at 1 min of life, but subsequent values were similar to 21% O(2) group between 2 and 30 min of life. Oxygen uptake from the lung and systemic oxygen extraction was similar between the two groups. Pulmonary arteries showed increased staining for superoxide anions and increased contractility to norepinephrine following resuscitation with 100% O(2). The increased PA contractility induced by 100% O(2) was reversed by scavenging superoxide anions with superoxide dismutase and catalase. We conclude that resuscitation of asphyxiated lambs with 100% O(2) increases Pa(O(2)) but does not improve lung oxygen uptake, decrease PVR at 30 min, or increase systemic oxygen extraction ratios. Furthermore, 100% O(2) also induces oxidative stress and increases PA contractility. These findings support the new neonatal resuscitation guidelines recommending 21% O(2) for initial resuscitation of asphyxiated neonates.     

Oxidative stress and antioxidant defenses in goldfish Carassius auratus during anoxia and reoxygenation

The purpose of this work was to evaluate the response of the antioxidant system of goldfish Carassius auratus during anoxia and reoxygenation. The exposure of goldfish to 8 h of anoxia induced a 14% decrease in total glutathione levels in the kidney, although the liver, brain, and muscle were unaffected. Anoxia also resulted in increases in the activities of liver catalase, brain glucose-6-phosphate dehydrogenase, and brain glutathione peroxidase (by 38, 26, and 79%, respectively) and a decrease in kidney catalase activity (by 17.5%). After 14 h of reoxygenation, liver catalase and brain glutathione peroxidase activities remained higher than controls and several other tissue-specific changes occurred in enzyme activities. Superoxide dismutase activity was unaffected by anoxia and reoxygenation. The levels of conjugated dienes, as indicators of lipid peroxidation, increased by 114% in liver after 1 h of reoxygenation and by 75% in brain after 14 h of reoxygenation. Lipid peroxidation was unaffected in kidney and depressed during anoxia and reoxygenation (by 44-61%) in muscle. Regulation of the goldfish antioxidant system during anoxia may constitute a biochemical mechanism that minimizes oxidative stress following reoxygenation.     

An overview of lipid peroxidation with emphasis in outer segments of photoreceptors and the chemiluminescence assay

The onset of lipid peroxidation within cellular membranes is associated with changes in their physicochemical properties and with the impairment of protein functions located in the membrane environment. This article provides current information on the origin and function of polyunsaturated fatty acids in nature, lipid peroxidation of cellular membranes: enzymatic (lipoxygenases) and non-enzymatic. The latest knowledge on in vivo biomarkers of lipid peroxidation including isoprostanes, isofurans and neuroprostanes are discussed. A further focus is placed on analytical methods for studying lipid peroxidation in membranes with emphasis in chemiluminescence and its origin, rod outer segments of photoreceptors, the effect of antioxidants, fatty acid hydroperoxides and lipid protein modifications. Since rhodopsin, the major integral protein of rod outer segments is surrounded by phospholipids highly enriched in docosahexaenoic acid, the author proposes the outer segments of photoreceptors as an excellent model to study lipid peroxidation using the chemiluminescence assay since these membranes contain the highest concentration of polyunsaturated fatty acids of any vertebrate tissue and are highly susceptible to oxidative damage.     

Molecular pharmacology of isoprostanes in vascular smooth muscle

Isoprostanes are marker of lipid peroxidation and are produced after free-radical attack of membrane lipids. In addition, they are biologically active and are essentially vaso- and broncho-constrictor. Their smooth muscle constrictor actions are closely linked to the activation of the thromboxane A(2) receptor, but also involve a distinct receptor not yet identified. The response of vascular smooth muscle to isoprostanes is subclass-specific (F-series versus E-series isoprostanes) and cell- and species-related. In this review, we will address the vascular actions of isoprostanes and their possible role in vascular physiology and pathophysiology.     

Plasma levels of soluble CD36, platelet activation, inflammation, and oxidative stress are increased in type 2 diabetic patients

Inflammation, oxidative stress, and platelet activation are involved in type 2 diabetes and its complications. Soluble CD36 (sCD36) has been proposed to early identify diabetics at risk of accelerated atherothrombosis. We aimed at characterizing the platelet contribution to sCD36 in diabetes, by correlating its concentration with the extent of platelet-mediated inflammation and in vivo lipid peroxidation and investigating the effects of low-dose aspirin on these processes. A cross-sectional comparison of sCD36, soluble CD40L (sCD40L) reflecting platelet-mediated inflammation, urinary 11-dehydro-TxB(2), and 8-iso-PGF(2α), in vivo markers of platelet activation and lipid peroxidation, was performed among 200 diabetic patients (94 of them on aspirin 100mg/day) and 47 healthy controls. sCD36 levels (median [IQR]: 0.72 [0.31-1.47] vs 0.26 [0.2-0.37], P=0.003) and urinary 11-dehydro-TxB(2) levels (666 [293-1336] vs 279 [160-396], P≤0.0001) were significantly higher in diabetic patients not on aspirin (n=106) than in healthy subjects. These variables were significantly lower in aspirin-treated diabetics than untreated patients (P<0.0001). Among patients not on aspirin, those with long-standing diabetes (>1 year) had significantly higher sCD36 levels in comparison to patients with diabetes duration <1 year (1.01 [0.62-1.86] vs 0.44 [0.22-1.21], P=0.001). sCD36 linearly correlated with sCD40L (rho=0.447; P=0.0001). On multiple regression analysis, 11-dehydro-TxB(2) (β=0.360; SEM=0.0001, P=0.001), 8-iso-PGF(2α) (β=0.469; SEM=0.0001, P<0.0001), and diabetes duration (β=0.244; SEM=0.207, P=0.017) independently predicted sCD36 levels. sCD36, platelet activation, inflammation, and oxidative stress are increased in type 2 diabetes. Future studies are needed to elucidate if the incomplete down-regulation of sCD36 by low-dose aspirin implies that sCD36 may be derived from tissues other than platelets or if additional antiplatelet strategies in diabetes are necessary to interrupt CD36-dependent platelet activation.