Isoprostanes: novel bioactive products of lipid peroxidation

Isoprostanes, are a novel group of prostaglandin-like compounds that are biosynthesised from esterified polyunsaturated fatty acid (PUFA) through a non-enzymatic free radical-catalysed reaction. Several of these compounds possess potent biological activity, as evidenced mainly through their pulmonary and renal vasoconstrictive effects, and have short half-lives. It has been shown that isoprostanes act as full or partial agonists through thromboxane receptors. Both human and experimental studies have indicated associations of isoprostanes and severe inflammatory conditions, ischemia-reperfusion, diabetes and atherosclerosis. Reports have shown that F₂-isoprostanes are authentic biomarkers of lipid peroxidation and can be used as potential in vivo indicators of oxidant stress in various clinical conditions, as well as in evaluations of antioxidants or drugs for their free radical-scavenging properties.

Higher levels of F₂-isoprostanes have been found in the normal human pregnancy compared to non-pregnancy, but their physiological role has not been well studied so far. Since bioactive F₂-isoprostanes are continuously formed in various tissues and large amounts of these potent compounds are found unmetabolised in their free acid form in the urine in normal basal conditions with a wide inter-individual variation, their role in the regulation of normal physiological functions could be of further biological interest, but has yet to be disclosed. Their potent biological activity has attracted great attention among scientists, since these compounds are found in humans and animals in both physiological and pathological conditions and can be used as reliable biomarkers of lipid peroxidation.     

Trace element,immune and opioid biomarkers of unstable angina,increased atherogenicity and insulin resistance: Results of machine learning

BackgroundAberrations in endothelial cells, immune and oxidative pathways are associated with atherosclerosis (ATS) and unstable angina (UA). The role of trace elements, minerals, and the endogenous opioid system (EOS) in UA are less well established.MethodsWe measured lipid, insulin resistance (IR), and immune, trace element (copper and zinc), mineral (magnesium, calcium), EOS (β-endorphin and mu-opioid receptor (MOR)) and antioxidant (vitamin D3) biomarkers in patients with ATS (n = 60) and UA (n = 60) and healthy controls (n = 58).ResultsATS patients showed increased atherogenic and IR indices, IL-6, IL-10, β-endorphin, copper and magnesium, and lower zinc than healthy controls. Logistic regression showed that UA was significantly discriminated from ATS without UA with an accuracy of 85.5 % using calcium, IL-10, β-endorphin, MOR, triglycerides, IR (all positively), and copper and vitamin D3 (inversely). Neural networks showed that UA was discriminated from ATS without UA with an area under the ROC curve of 0.942 using MOR, β-endorphin, calcium, insulin resistance, vitamin D3 and copper as input variables. We found that 50.0 % of the variance in IR was explained by the regression on copper, IL-10, IL-6 (all positively), and zinc (inversely), while 32.9 % of the variance in the atherogenic index of plasma was explained by copper, IL-10 (both positively), and magnesium (inversely).ConclusionUA is not only mediated by insulin resistance, atherogenicity, and immune disorders, but also by aberrations in the endogenous opioid system and trace elements as well as lowered antioxidant levels. Copper appears to play a key role in IR and atherogenicity.     

Measurement of Prostaglandins in the Cerebrospinal Fluid in Cat, Dog, and Man

Prostaglandins are involved in the modulation of various central functions (neurotransmitters and hypothalamic hormone release, thermoregulation, cerebro-vascular tone) and their levels increase in pathological situations [subarachnoid hemorrhage (SAH), stroke, convulsive disorders, etc.]. This study, using sensitive and specific antibodies, examined levels of four eicosanoids, Prostaglandins E₂ and F_2α(PGE₂, PGF_2α); and the metabolites of PGI₂, 6-keto-prostaglandin F_1α (6-keto-PGF1_α) and of thromboxane A₂, thromboxane B₂ (TxB₂), in the cerebrospinal fluid (CSF) obtained atraumatically from three species (human, canine, and feline). An assessment of the methodologic procedures (extraction and radioimmunoassay) was carried out. Human lumbar cerebrospinal fluid was shown to contain PGF_2α (15–44 pg/ml), 6-keto-PGF_1α (undetectable to 39 pg/ml), and TxB₂ (un-detectable to 28 pg/ml), whereas PGE₂ was undetectable (>18 pg) in all cases. In both animals species the eico-sanoid concentrations were 3-to 30-fold higher than humans for every prostaglandin examined. Although the prostaglandin profile for a given species remained constant (cat, PGE₂:6-keto-PGF_1α:TxB₂:PGF_2α; dog, TxB₂:PGE₂:6-keto-PGF_1α:PGF_2α), the absolute levels were found to be lower in the pentobarbital-anesthetized animals than in conscious cats. The correspondence of the prostaglandin profiles found in cerebrospinal fluid with the profiles reported in the literature in brain homogenates for the same species supports the hypothesis that cerebrospinal fluid levels of prostaglandins reflect the relative rates of synthesis in neural tissue.     

The relationship between oxidative stress and nonalcoholic fatty liver disease: Its effects on the development of nonalcoholic steatohepatitis

Abstract

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are the most common underlying causes of chronic liver injury. They are associated with a wide spectrum of hepatic disorders including basic steatosis, steatohepatitis, and cirrhosis. The molecular and cellular mechanisms underlying hepatic injury in NAFLD and NASH are still unknown. This review describes the roles of oxidative stress and inflammatory responses in the pathogenesis of NAFLD and its progression to NASH.     

Normal or stress-induced fibroblast senescence involves COX-2 activity

Cyclooxygenase-2 (COX-2) is an inducible enzyme of the prostaglandin biosynthesis pathway. It is involved in many stress responses, and its activity can produce oxidative damage, suggesting it could participate in senescence. In this study, COX-2 expression is shown to increase during senescence of normal human dermal or prostatic fibroblasts, and the ensuing prostaglandin E(2) (PGE(2)) production to increase about 10-fold. Enhancing this COX-2 activity by supplying exogenous arachidonic acid accelerates the occurrence of the major markers of senescence, cell-size increase, spreading, senescence-associated-beta-galactosidase (SA-beta-Gal) activity and growth plateau. Conversely, blocking this COX-2 activity with the specific inhibitor NS398 partially inhibited the occurrence of these markers. COX-2 expression and PGE(2) production are also increased about 10-fold during both NF-kappaB- or H(2)O(2)-induced senescence. Using NS398 or small interferent RNA specifically targeting COX-2 attenuated the appearance of the SA-beta-Gal activity and growth arrest in both stress situations. Taken together, these findings indicate that COX-2 is highly up-regulated during both normal and stress-induced fibroblast senescence and contributes to the establishment of the senescent characteristics.     

Both cyclooxygenase- and cytokine-mediated inflammation are associated with carotid intima-media thickness

BACKGROUND: Common carotid artery intima-media thickness (CCA-IMT) is a valid index of atherosclerosis, which is viewed as an inflammatory disease. It is unknown if various modes of inflammation (cyclooxygenase [COX]-mediated, cytokine-mediated), oxidative stress and anti-oxidants are independently related to CCA-IMT. METHODS AND RESULTS: We investigated cross-sectional relations between CCA-IMT measured by B-mode ultrasound and COX-mediated inflammation (as measured by 15-keto-dihydro-prostaglandin F(2alpha) [PGF(2alpha)], cytokine-mediated inflammation (interleukin-6 [IL-6], high sensitivity C-reactive protein [hsCRP] and serum amyloid A protein [SAA]), oxidative stress (8-iso-PGF(2alpha), an F(2)-isoprostane; a non-enzymatic, free radical-induced product of arachidonic acid), and tocopherols (anti-oxidants) in a small subset of a population-based sample of elderly men (n=234) stating no use of anti-inflammatory medications. In a backward-stepwise regression analysis of correlates of CCA-IMT (with PGF(2alpha), hsCRP, IL-6, SAA, F(2)-isoprostanes, tocopherols, diabetes, body mass index (BMI), beta-blocker, statin treatment, smoking, hypertension and cholesterol), PGF(2alpha), CRP, beta-blocker treatment, diabetes and BMI were independently associated with CCA-IMT. There were no associations between F(2)-isoprostanes or tocopherols and CCA-IMT in this study. CONCLUSION: This study suggests both COX- and cytokine-mediated inflammation to be independently associated with increased CCA-IMT, implying that there might be more than one mode of inflammation involved in atherogenesis.     

Prejunctional Inhibitory Effects of Isoprostanes on Dopaminergic Neurotransmission in Bovine Retinae,In vitro

We investigated the effect of isoprostanes (IsoPs) on potassium (K⁺)-depolarization-evoked release of [³H]dopamine from isolated bovine retinae. Isolated retinae were preloaded with [³H]dopamine and then prepared for studies of [³H]dopamine release using the superfusion method. 8-iso(15R)PGF_2α, 8-isoPGE₂, 8-isoPGE₁ and 8-isoPGF_2α attenuated [³H]dopamine release from isolated bovine retinae. At a concentration of 1 μM, the rank order of activity displayed by IsoP agonists was: 8-iso(15R)PGF_2α > 8-isoPGE₂ > 8-isoPGE₁ > 8-isoPGF_2α. Inhibition of cyclooxygenase (COX) with flurbiprofen reversed the effects caused by 8-isoPGE₂ (10 nM and 10 μM), 8-iso(15R)PGF_2α (1 μM) and 8-isoPGE₁ (1 μM). Although the EP1/EP2 antagonist, AH 6809 (10 μM) had no significant effect on K⁺-induced [³H]dopamine release, it blocked the inhibitory effect of both 8-isoPGE₁ (10 μM) and 8-isoPGE₂ (10 μM). In conclusion, IsoPs attenuate K⁺-induced [³H]dopamine release in isolated bovine retinae, presumably via an indirect action on COX pathway leading to the production of prostanoids, which in turn, activates EP receptors.     

Peroxisomes, oxidative stress, and inflammation

Peroxisomes are intracellular organelles mediating a wide variety of biosynthetic and biodegradative reactions.Included among these are the metabolism of hydrogen peroxide and other reactive species,molecules whose levels help define the oxidative state of cells.Loss of oxidative equilibrium in cells of tissues and organs potentiates inflammatory responses which can ultimately trigger human disease.The goal of this article is to review evidence for connections between peroxisome function,oxidative stress,and inflammation in the context of human health and degenerative disease.Dysregulated points in this nexus are identified and potential remedial approaches are presented.     

Antiinflammatory effects of the cyclopentenone isoprostane 15-A(2)-IsoP in human gestational tissues

Proinflammatory prostaglandins and cytokines are involved in the initiation of human labor and delivery. Although cyclopentenone prostaglandins regulate the formation of these prolabor mediators via nuclear factor-κB (NF-κB) and/or peroxisome proliferator-activated receptor-γ, recent evidence suggests that they do not exist in vivo. Cyclopentenone isoprostanes (IsoPs), which are highly reactive structural isomers of bioactive cyclopentenone prostaglandins, do exist physiologically and have been shown to inhibit the inflammatory response in macrophages. Therefore the aim of this study was to determine the effect of the synthetic cyclopentenone IosP 15-A₂-IsoP on the expression of prolabor mediators in human gestational tissues. Human placenta and gestational membranes (n = 5) were incubated in the absence or presence of 12.5, 25, and 50 μM 15-A₂-IsoP with 10 μg/ml lipopolysaccharide (LPS). Treatment of placenta and fetal membranes with 15-A₂-IsoP caused a dose-dependent decrease in LPS-stimulated release of the cytokines IL-1β, IL-6, IL-8, and TNF- and the prostaglandins PGE₂ and PGF₂. NF-κB p65 DNA binding activity was significantly inhibited by treatment with 50 μM 15-A₂-IsoP. Collectively, these data suggest that 15-A₂-IsoP exhibits antiinflammatory properties via antagonism of NF-κB activity. Cyclopentenone IsoPs may serve as negative feedback regulators of the inflammatory response in human gestational tissues.     

Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa

Two metal response elements, flanking an antioxidant response element, were identified in regions upstream (-3730 bp) to copper metallothionein (CuMT) gene of Neurospora crassa. Presence of copper in culture media, but not of pro-oxidants like H2O2 or menadione, induced CuMT gene expression that could not be completely abolished by antioxidants such as N-acetyl cysteine and ascorbic acid. Gel shift assays revealed the ability of nuclear extracts from copper induced cultures to bind PCR-amplified metal response or antioxidant response elements. Similar observations could not be made with cultures exposed either to pro-oxidants or antioxidants. These results differentiate between CuMT gene induction by copper from antioxidant functions associated with the identified upstream elements.     

Towards the prevention of potential aluminum toxic effects and an effective treatment for Alzheimer's disease

In 1991, treatment with low dose intramuscular desferrioxamine (DFO), a trivalent chelator that can remove excessive iron and/or aluminum from the body, was reported to slow the progression of Alzheimer's disease (AD) by a factor of two. Twenty years later this promising trial has not been followed up and why this treatment worked still is not clear. In this critical interdisciplinary review, we provide an overview of the complexities of AD and involvement of metal ions, and revisit the neglected DFO trial. We discuss research done by us and others that is helping to explain involvement of metal ion catalyzed production of reactive oxygen species in the pathogenesis of AD, and emerging strategies for inhibition of metal-ion toxicity. Highlighted are insights to be considered in the quests to prevent potentially toxic effects of aluminum toxicity and prevention and intervention in AD.     

The isoprostanes: Unique bioactive products of lipid peroxidation

The development of a specific, reliable and noninvasive method for measuring oxidative stress in humans is essential for establishing the role of free radicals in human diseases. Currently, accurate techniques to assess oxidant injury in vivo are extremely limited although a number of approaches are being investigated. Of these, the measurement of specific products of nonenzymatic lipid peroxidation, the F₂-isoprostanes (F₂-IsoPs), appears to be a more accurate marker of oxidative stress in vivo in humans than other available methods. The purpose of this brief review is to acquaint the reader with the IsoPs from a biochemical perspective and to provide information regarding the utility of quantifying these compounds as indicators of oxidant stress.     

Quantification of dinor, dihydro metabolites of F2-isoprostanes in urine by liquid chromatography/tandem mass spectrometry

The F2-isoprostanes (F2-IsoP) are a series of prostaglandin (PG)-F2-like compounds that are produced by free-radical-mediated oxidation of arachidonic acid. One F2-IsoP with potent biological activity is 15-F2t-IsoP and increased levels of 15-F(2t)-IsoP have been measured in several diseases. The major urinary metabolite of 15-F2t-IsoP (8-iso-PGF(2alpha)) is 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M). Previously, we developed a stable isotope dilution gas chromatography/negative chemical ionization/mass spectrometry (MS) assay for 15-F2t-IsoP-M, which, while highly sensitive, required time-consuming derivatization and thin-layer chromatography purification. We now report the development of a more rapid high-performance liquid chromatography method coupled to electrospray ionization-tandem mass spectrometry (LC/MS/MS) to analyze all of the dinor,dihydro metabolites of the F2-IsoP isomers (F2-IsoP-M). The precision of this assay was +/-5.0% and the accuracy 80%. The assay remained linear over a range of 1-100 ng injected onto the LC column. Levels of F2-IsoP-M determined by the LC/MS/MS assay method significantly correlated with levels of 15-F2t-IsoP-M determined by the GC/MS assay (R = 0.77y = 67.2x-0.5). The levels of F2-IsoP-M detected in spot urines from 40 normal subjects were 38.1+/-19.1 ng/mg creatinine (mean+/-SD). This method provides an accurate and rapid assay to assess oxidative status in vivo.     

抗氧化剂在糖尿病中的应用研究进展

氧化应激是自由基的促氧化与机体的抗氧化失衡造成的.自由基的高反应活性可以使细胞组分发生化学变化,并导致脂质过氧化.越来越多的证据表明:糖尿病患者体内活性氧物质明显增多,并且抗氧化防御系统功能紊乱.抗氧化剂可以降低糖尿病患者体内的氧化应激水平,清除自由基并改善抗氧化防御体系,所以将抗氧化剂应用于糖尿病是可行的.许多抗氧化剂已经用于糖尿病的研究与治疗,本文主要将作用于糖尿病的各种抗氧化剂做一综述.     

Porous graphitic carbon chromatography-tandem mass spectrometry for the study of isoprostanes in human cerebrospinal fluid

F2-isoprostanes are produced by the non-enzymatic peroxidation of arachidonic acid in membrane phospholipids. This paper describes a new method for the determination of all four classes of F2-isoprostanes in human cerebrospinal fluid (CSF) involving separation on a 1 mm x 150 mm porous graphitic carbon (PGC) column and detection by triple quadrupole mass spectrometry in negative-ion electrospray mode. The sample pre-treatment consisted of an ultrafiltration step, following which 300 microl of CSF sample could be injected directly onto a 1 mm x 10 mm PGC guard column functioning as a trap for the analytes. The loading solvent was Milli-Q water at 125 microl/min. After 3 min, the sample was switched into the separation column. The F2-isoprostanes were separated in 20 min using a linear solvent gradient comprising water, methanol, acetonitrile and ammonium hydroxide at a pH of 9.5 and a flow of 50 microl/min The limit of detection (calculated as 3S/N) was approximately 40 pM (14 pg/ml). The assay was linear within the examined range (18-450 pg/ml), using CSF spiked with iPF2alpha-III standard (r(2)>0.995). Repeatability data were calculated for CSF spiked to 90 pg/ml and the relative standard deviation (RSD) obtained was 3% (n=6).     

Role of antioxidants in protecting cellular DNA from damage by oxidative stress

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H₂O₂ was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H₂O₂ scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 μM Zn²⁺ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H₂O₂ suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.