Quantification of F-ring isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay

Lipid peroxidation has been implicated in the pathophysiological sequelae of human neurodegenerative disorders. It is recognized that quantification of lipid peroxidation is best assessed in vivo by measuring a series of prostaglandin (PG) F2-like compounds termed F2-isoprostanes (IsoPs) in tissues in which arachidonic acid is abundant. Unlike other organs, the major polyunsaturated fatty acid (PUFA) in the brain is docosahexaenoic acid (DHA, C22:6 omega-6), and this fatty acid is particularly enriched in neurons. We have previously reported that DHA undergoes oxidation in vitro and in vivo resulting in the formation of a series of F2-IsoP-like compounds termed F4-neuroprostanes (F4-NPs). We recently chemically synthesized one F4-NP, 17-F4c-NP, converted it to an 18O-labeled derivative, and utilized it as an internal standard to develop an assay to quantify endogenous production of F4-NPs by gas chromatography (GC)/negative ion chemical ionization (NICI) mass spectrometry (MS). The assay is highly precise and accurate. The lower limit of sensitivity is approximately 10 pg. Levels of F4-NPs in brain tissue from rodents were 8.7 +/- 2.0 ng/g wet weight (mean +/- S.D.). Levels of the F4-NPs in brains from normal humans were found to be 4.9 +/- 0.6 ng/g (mean +/- S.D.) and were 2.1-fold higher in affected regions of brains from humans with Alzheimer's disease (P = 0.02). Thus, this assay provides a sensitive and accurate method to assess oxidation of DHA in animal and human tissues and will allow for the further elucidation of the role of oxidative injury to the central nervous system in association with human neurodegenerative disorders.     

Vitamin E supplementation and plasma 8-isoprostane and adiponectin in overweight subjects

Objective: Isoprostanes are a marker of oxidant stress and atherosclerotic risk, and plasma concentrations are elevated in obesity. Adiponectin is a regulator of insulin sensitivity, and low circulating levels are associated with oxidant stress and obesity. The aim of this study was to determine the effect of vitamin E supplementation on plasma concentrations of 8‐isoprostane and adiponectin in overweight/obese subjects. Research Methods and Procedures: The study was a 6‐month, randomized, double‐blind, placebo‐controlled trial in 80 overweight subjects (60 women and 20 men, BMI >27 kg/m²). Exclusion criteria were serious illness, smoking, or taking antioxidant supplements. Participants were randomized to receive 800 IU/d natural vitamin E (n = 39) or placebo (n = 41) for 3 months with an increase in the dose to 1200 IU/d for a further 3 months. Plasma 8‐isoprostane and adiponectin concentrations were measured at baseline and 3 and 6 months. Results: During 6 months of supplementation with vitamin E, plasma vitamin E concentration increased significantly (p < 0.001) by 76%, and plasma 8‐isoprostane concentrations decreased significantly (?11%, p = 0.03), whereas plasma adiponectin concentrations did not change significantly. Discussion: These findings suggest that supplementation with high‐dose vitamin E decreases systemic oxidative stress and 8‐isoprostane concentrations in overweight/obese individuals. A decrease in plasma 8‐isoprostane has the potential to reduce risk of cardiovascular disease in obesity.     

Impairment of 8-iso-PGF2ALPHA isoprostane metabolism by dietary conjugated linoleic acid (CLA)

8-iso-PGF_2α isoprostane (IP) is one of the most-used markers of lipid peroxidation in experimental models and humans. After its formation, it is promptly metabolized to 2,3 dinor (DIN) in peroxisomes.Conjugated linoleic acid (CLA) is preferentially β-oxidized in peroxisomes which may compete with IP, and thereby may affect its metabolism.In order to verify whether CLA is able to influence IP formation and/or metabolism and to explain the mechanism, we challenged rats supplemented with CLA or with triolein (as a control fatty acid), with a single dose of carbon tetrachloride (CCl₄) or of bacterial lipopolysaccharide (LPS). The results showed that IP and its precursor arachidonic acid hydroperoxide, as well as malondialdheyde (MDA), increase significantly in the liver of rats challenged with CCl₄, irrespective of the diet, while in LPS-treated rats only nitrites in liver and isoprostane in plasma increase. On the other hand, the peroxisomal β-oxidation products of IP, the DIN, is significantly lower in the CLA group with respect to control and triolein groups.To further investigate whether this is due to competition between CLA and IP at the cellular level, we incubated human fibroblasts from healthy subjects or patients with adrenoleukodystrophy (ALD), with CLA and/or commercially available IP. The rationale of this approach is based on the deficient peroxisomal β-oxidation of fibroblasts from ALD patients, leading to a reduced formation of DIN. In both normal and ALD cells, the presence of CLA significantly inhibits the formation of DIN from IP.We may conclude that both in vitro and in vivo studies strongly suggest that CLA may impair IP catabolism in peroxisomes. Consequently an increase of IP, as a sole result of CLA intake, cannot be considered as a marker of lipid peroxidation.     

Cyclo-oxygenase-1 and -2 differently contribute to prostaglandin E2 synthesis and lipid peroxidation after in vivo activation of N-methyl-D-aspartate receptors in rat hippocampus

Using intracerebral microdialysis, we reported previously that acute in vivo activation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) and F2-isoprostane 15-F(2t)-IsoP in the hippocampus of freely moving rats. The formation of the two metabolites--produced through cyclo-oxygenase (COX) enzymatic activity and free radical-mediated peroxidation of arachidonic acid (AA), respectively,--was prevented by the specific NMDA antagonist MK-801, and was largely dependent on COX-2 activity. Here, we demonstrate that besides COX-2, which is the prominent COX isoform in the brain and particularly in the hippocampus, the constitutive isoform, COX-1 also contributes to prostaglandin (PG) synthesis and oxidative damage following in vivo acute activation of hippocampal NMDA glutamate receptors. The relative contribution of the two isoforms is dynamically regulated, as the COX-2 selective inhibitor NS398 immediately prevented PGE2 and 15-F(2t)-IsoP formation during the application of NMDA, whereas the COX-1 selective inhibitor SC560 was effective only 1 h after agonist infusion. Our data suggest that, although COX-2 is the prominent isoform, COX-1 activity may significantly contribute to excitotoxicity, particularly when considering the amount of lipid peroxidation associated with its catalytic cycle. We suggest that both isoforms should be considered as possible therapeutic targets to prevent brain damage caused by excitotoxicity.     

Lung cancer biomarkers for the assessment of modified risk tobacco products: an oxidative stress perspective

Abstract

Manufacturers have developed prototype cigarettes yielding reduced levels of some tobacco smoke toxicants, when tested using laboratory machine smoking under standardised conditions. For the scientific assessment of modified risk tobacco products, tests that offer objective, reproducible data, which can be obtained in a much shorter time than the requirements of conventional epidemiology are needed. In this review, we consider whether biomarkers of biological effect related to oxidative stress can be used in this role. Based on published data, urinary 8-oxo-7,8-dihydro-2-deoxyguanosine, thymidine glycol, F₂-isoprostanes, serum dehydroascorbic acid to ascorbic acid ratio and carotenoid concentrations show promise, while 4-hydroxynonenal requires further qualification.     

Oxidative stress early in pregnancy and pregnancy outcome

The objectives of this study were to determine whether oxidative stress early in pregnancy influenced pregnancy outcome. A combination of assays were used for exogenous and endogenous anti-oxidants together with two well accepted biomarkers for oxidative stress, the urinary excretion of 8-iso-PGF_2α (a biomarker marker for lipid oxidation, n=508) and 8-oxo-7,8 dihydro-2 deoxyguanosine (8-OHdG, a biomarker for DNA oxidation, n=487). The two biomarkers tracked different pregnancy outcomes. Isoprostanes were associated with an increased risk of pre-eclampsia and a decreased proportion of female births. In contrast, 8-OHdG tracked lower infant birthweight and shortened gestation duration. Birth defects were associated with low levels of 8-OHdG.     

Evaluation of urinary biomarkers of oxidative/nitrosative stress in adolescents and adults with Down syndrome

Urinary biomarkers of oxidative stress have been little studied in adults with Down syndrome (DS), usually no more than two biomarkers have been measured in the population studied and controversial results are reported in literature. Thus, we aimed to assess a set of oxidative and nitrosative stress biomarkers in urine samples of adolescents and adults with DS, with and without hypothyroidism, which comprise: 8-hydroxy-2′-deoxyguanosine (8-OHdG), isoprostane 15-F_2t-IsoP, thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs), dityrosine (diTyr), hydrogen peroxide (H₂O₂) and nitrite/nitrate (NOx). Fluorimetric and spectrophotometric assays were performed in DS (n = 78), some of them taking levothyroxine for hypothyroidism (n = 24), and in their healthy age-matched controls (n = 65). We found that levels of AGEs, diTyr, H₂O₂ and NOx are increased in DS patients in any or in all age groups, whereas Cr levels were lower in DS than in controls in all age groups. Besides, correlations with age in DS were positive for diTyr and negative for Cr, TBARS, 15-F_2t-IsoP and NOx. We also found lower levels of Cr from 15 to 19 years, higher levels of TBARS and AGEs from 20 to 40 years and higher levels of diTyr from 15 to 40 years in DS patients receiving levothyroxine than in DS without hypothyroidism diagnosed. We conclude that AGEs, diTyr, H₂O₂ and NOx could be used as oxidative stress biomarkers in DS in contrast to 8-OHdG, 15-F_2t-IsoP and TBARS, at least with the methods used. However, renal impairment could occur in DS and Cr adjustment may bias the results, particularly in hypothyroid patients.     

Aging, gender and APOE isotype modulate metabolism of Alzheimer's Abeta peptides and F-isoprostanes in the absence of detectable amyloid deposits

Aging and apolipoprotein E (APOE) isoform are among the most consistent risks for the development of Alzheimer's disease (AD). Metabolic factors that modulate risk have been elusive, though oxidative reactions and their by-products have been implicated in human AD and in transgenic mice with overt histological amyloidosis. We investigated the relationship between the levels of endogenous murine amyloid beta (Abeta) peptides and the levels of a marker of oxidation in mice that never develop histological amyloidosis [i.e. APOE knockout (KO) mice with or without transgenic human APOEepsilon3 or human APOEepsilon4 alleles]. Aging-, gender-, and APOE-genotype-dependent changes were observed for endogenous mouse brain Abeta40 and Abeta42 peptides. Levels of the oxidized lipid F2-isoprostane (F2-isoPs) in the brains of the same animals as those used for the Abeta analyses revealed aging- and gender-dependent changes in APOE KO and in human APOEepsilon4 transgenic KO mice. Human APOEepsilon3 transgenic KO mice did not exhibit aging- or gender-dependent increases in F2-isoPs. In general, the changes in the levels of brain F2-isoPs in mice according to age, gender, and APOE genotype mirrored the changes in brain Abeta levels, which, in turn, paralleled known trends in the risk for human AD. These data indicate that there exists an aging-dependent, APOE-genotype-sensitive rise in murine brain Abeta levels despite the apparent inability of the peptide to form histologically detectable amyloid. Human APOEepsilon3, but not human APOEepsilon4, can apparently prevent the aging-dependent rise in murine brain Abeta levels, consistent with the relative risk for AD associated with these genotypes. The fidelity of the brain Abeta/F2-isoP relationship across multiple relevant variables supports the hypothesis that oxidized lipids play a role in AD pathogenesis, as has been suggested by recent evidence that F2-isoPs can stimulate Abeta generation and aggregation.     

Multiple Actions of the Human Immunodeficiency Virus Type-1 Tat Protein on Microglial Cell Functions

The human immunodeficiency virus type-1 (HIV-1) regulatory protein Tat is produced in the early phase of infection and is essential for virus replication. Together with other viral products, Tat has been implicated in the pathogenesis of HIV-1-associated dementia (HAD). As HIV-1 infection in the brain is very limited and macrophage/microglial cells are the only cellular type productively infected by the virus, it has been proposed that many of the viral neurotoxic effects are mediated by microglial products. We and others have shown that Tat affects the functional state of microglial cells, supporting the hypothesis that activated microglia play a role in the neuropathology associated with HIV-1 infection. This review describes the experimental evidence indicating that Tat stimulates microglia to synthesize potentially neurotoxic molecules, including proinflammatory cytokines and free radicals, and interferes with molecular mechanisms controlling cAMP levels, intracellular [Ca2+], and ion channel expression.     

Biomarkers of lipid peroxidation in clinical material

Background

Free radical-mediated lipid peroxidation has been implicated in a number of human diseases. Diverse methods have been developed and applied to measure lipid peroxidation products as potential biomarkers to assess oxidative stress status in vivo, discover early indication of disease, diagnose progression of disease, and evaluate the effectiveness of drugs and antioxidants for treatment of disease and maintenance of health, respectively. However, standardized methods are not yet established.

Scope of review

Characteristics of various lipid peroxidation products as biomarkers are reviewed on the basis of mechanisms and dynamics of their formation and metabolism and also on the methods of measurement, with an emphasis on the advantages and limitations.

Major conclusions

Lipid hydroxides such as hydroxyoctadecadienoic acids (HODE), hydroxyeicosatetraenoic acids (HETE), and hydroxycholesterols may be recommended as reliable biomarkers. Notably, the four HODEs, 9-cis,trans, 9-trans,trans, 13-cis,trans, and 13-trans,trans-HODE, can be measured separately by LC–MS/MS and the trans,trans-forms are specific marker of free radical mediated lipid peroxidation. Further, isoprostanes and neuroprostanes are useful biomarker of lipid peroxidation. It is important to examine the distribution and temporal change of these biomarkers.

General significance

Despite the fact that lipid peroxidation products are non-specific biomarkers, they will enable to assess oxidative stress status, disease state, and effects of drugs and antioxidants. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.