Characterization of 8-epiprostaglandin F2alpha as a marker of amyloid beta-peptide-induced oxidative damage

The amyloid beta-peptide (A beta) is a major component of the neuritic plaques that are a defining histological characteristic of Alzheimer's disease. A beta can be directly toxic and pro-inflammatory to cells in vitro. Numerous reports have shown that oxidative damage and reactive oxygen species play a role in A beta-mediated neurotoxicity. 8-Epiprostaglandin F2alpha (8-isoprostane) is a well characterized product of lipid peroxidation that is formed nonenzymatically in cell membranes following an oxidative insult. We report a time- and concentration-dependent increase in 8-isoprostane levels in rat hippocampal cultures treated with A beta(1-40) or hydrogen peroxide. As evidence that 8-isoprostane production is part of an A beta toxic pathway, alkaline-treated peptide, which shows minimal toxic activity, resulted in greatly attenuated 8-isoprostane production. Although the increase in 8-isoprostane levels preceded cell death, exogenously added 8-isoprostane had no cytotoxic effects. The antioxidants vitamin E and propyl gallate attenuated A beta-induced 8-isoprostane formation yet had no effect on A beta-induced lactate dehydrogenase release. Neither vitamin E nor propyl gallate had any effect on A beta's ability to adopt a beta-pleated sheet structure and deposit on cells as determined by thioflavine S fluorescence. We conclude that 8-isoprostane is an indicator of A beta-induced damage but not necessarily a mediator of A beta-induced neurotoxicity. Also, 8-isoprostane could be a useful marker for assessing oxidative damage in the CNS.     

Identification of 8-iso-prostaglandin F(2alpha) in rat brain neuronal endings: a possible marker of membrane phospholipid peroxidation

Isoprostanes are a family of prostaglandin (PG) F and E isomers generated by free-radical attack from membrane bound arachidonic acid. We measured detectable levels of 8-iso-PGF(2alpha) in the perfusates of synaptosomes obtained from different areas of the rat brain cortex. A small but significant release of this isoprostane was found under basal conditions from all the areas explored; being lower in the dorsal cortex in respect to the frontal, parietal and occipital areas. Exposure of synaptosomes to a phospholipase A(2) activator, i.e. calcium-ionophore A23187, an oxidant agent, such as hydrogen peroxide or amyloid beta-peptide did not modify 8-iso-PGF(2alpha) release when these stimuli were applied separately. However, either hydrogen peroxide or amyloid beta-peptide increased 8-iso-PGF(2alpha) release in a dose-dependent manner, when given in the presence of the calcium-ionophore A23187. Synaptosome treatment with a non-selective cyclooxygenase inhibitor (fenoprofen) did not modify 8-iso-PGF(2alpha) release in any way, but treatment with a water soluble antioxidant (Trolox C) completely suppressed isoprostane release under basal conditions, as well as after the oxidant injury induced either by hydrogen peroxide or amyloid beta-peptide. We conclude that, in neuronal endings, 8-iso-PGF(2alpha) is generated under basal conditions and its formation may be increased in a dose-dependent fashion by oxidant stimuli through a cyclooxygenase-independent mechanism involving free radical-catalyzed oxidation of arachidonic acid on membrane phospholipids.     

Increased urinary excretion of 8-iso-prostaglandin F2alpha in patients with HFE-related hemochromatosis: a case-control study

The hypothesis according to which iron overload could be harmful has been extensively and controversially discussed in the literature. One underlying pathological mechanism may be elevated oxidative stress. Thus, we studied the correlation between hemochromatosis and an established marker of oxidative stress, 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha, iPF2alpha-III, 15-F2t-IsoP). We enrolled 21 patients with hemochromatosis, positive for the homozygous C282Y mutation in the HFE gene, and 21 healthy controls frequency-matched by age and gender in a case-control study design. The objective was to show that iron overload in HFE-related hemochromatosis is associated with increased oxidative stress assessed through 8-iso-PGF(2alpha) urinary excretion, and that oxidative stress is impacted by iron-removal treatment (phlebotomy). Study parameters were transferrin saturation, 8-iso-PGF(2alpha) urine excretion, transferrin, ferritin, serum iron, and vitamins A and E for all participants. Iron concentration in the liver and non-transferrin-bound iron were measured in patients only. We found a significant difference in 8-iso-PGF2alpha in patients (245 [interquartile range 157-348] pg/mg creatinine) compared with controls (128 [106-191] pg/mg creatinine, P = 0.002). Vitamin A was significantly reduced in cases (0.34 [0.25-1.83] microg/ml compared to 3.00 [2.11-3.39] microg/ml, P < 0.001), while vitamin E did not show a significant difference in cases (14.7 [11.5-18.1] microg/ml) compared with controls (14.9 [13.1-19.2] microg/ml, P = 0.52). After phlebotomy treatment and normalization of the iron parameters in the hemochromatosis group, serum vitamin A levels were significantly increased (1.36 [1.08-1.97] microg/ml, P = 0.035 vs. baseline, P < 0.001 vs. controls) and 8-iso-PGF2alpha urinary excretion was lowered to control levels (146 [117-198] pg/mg creatinine, P = 0.38 vs. controls). In our study, HFE-related hemochromatosis was associated with increased oxidative stress and hypovitaminemia A in C282Y homozygotes. The increased oxidative stress was reversible by normalization of the iron load by phlebotomy. Thus, phlebotomy is an effective and adequate means for reducing oxidative stress in these patients.     

Urinary levels of 8-hydroxydeoxyguanosine as a marker of oxidative damage in road cycling

We have determined the urinary 8-hydroxydeoxyguanosine (8-OHdG) levels of eight professional cyclists during a 4-day and a 3-week stage races. Monitoring of heart rates was used to establish zones corresponding to different intensities of exercise. The urinary 8-OHdG excretion, expressed by body weight, increased significantly in the first day or the first week of each race, respectively, and did not show further increases thereafter. Maximum 8-OHdG levels were reached in parallel to longer times spent at high intensities of exercise. Urinary excretion of creatinine increased with exercise, and changes in 8-OHdG levels were not detected when corrected by creatinine excretion. Serum glutathione concentrations did not change significantly at any point during exercise. We conclude that road cycling courses with an oxidative damage to DNA, which is sustained as long as the exercise is repeated. Both adaptation of antioxidant defenses and a decreased capacity to maintain a high intensity of effort may contribute to explain the absence of progressive increases in 8-OHdG excretion. The results of this study also confirm that the correction procedure using the amount of creatinine excreted should not be used when studying effects of exercise on urinary 8-OHdG.     

Spontaneous oxidative DNA damage in bovine retained and nonretained placental membranes

Retention of fetal membranes (RFM) is believed to be associated with conditions of oxidative stress. In this study, 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was used for the determination of spontaneous oxidative DNA lesions in maternal and fetal parts of bovine retained and nonretained placentas. Placental specimens were collected directly after spontaneous delivery or during cesarean section from cows divided into 6 groups: (A) cesarean section before term without RFM, (B) with RFM, (C) cesarean section at term without RFM, (D) with RFM, (E) spontaneous delivery at term without RFM and (F) with RFM. Isolated DNA was hydrolyzed and analyzed by HPLC; native nucleosides were monitored at 254 nm and 8-OH-dG by electrochemical detection. No significant differences in 8-OH-dG levels between retained and nonretained placental tissues were found in all samples from preterm groups (mean concentrations between 13 and 42 micromol/mol deoxyguanosine (dG)). In the term cesarean section group with RFM a significant increase in 8-OH-dG concentration in DNA from maternal (8-fold) and fetal (18-fold) membranes were detected when compared to the respective nonretained tissues. Also, in the term spontaneous delivery groups maternal nonretained placental tissues showed increased levels of 8-OH-dG in comparison to the respective tissues of the retained placenta group. In placental tissues oxidative DNA lesions appear to be controlled by responsive mechanisms which, possibly following exhaustion, give rise to increased 8-OH-dG levels.     

Comparison of different methods of measuring 8-oxoguanine as a marker of oxidative DNA damage

We are attempting to resolve some of the problems encountered in measuring 8-hydroxy-2′-deoxyguanosine (8-oxodG) in human cellular DNA as a marker of oxidative stress. Samples of authentic 8-oxodG were distributed, and participating laboratories undertook to analyse this material within a specified period. Most HPLC procedures gave values for 8-oxodG within ±40% of the target, as did two of four GC-MS procedures, and both LC-MS-MS methods. Calf thymus DNA samples containing increasing amounts of 8-oxodG were also distributed for analysis. Fewer than half the procedures tested were able to detect the dose response; those that were successful tended to be procedures with low coefficients of variation. For the analysis of 8-oxodG in human cells, where it is likely to be present at much lower concentrations than in the calf thymus DNA, it is crucial to reduce analytical variation to a minimum; a coefficient of variation of less than 10% should be the aim, to give reasonable precision. HPLC with amperometric electrochemical detection is not recommended, as it is less sensitive than coulometric detection. Immunological detection, ³²P-postlabelling and LC-MS-MS are alternative approaches to measurement of 8-oxodG in DNA that, on the grounds of precision and detection of dose response, cannot at present be recommended.     

Prostaglandin E(2) 9-keto reductase activity in bovine retained and not retained placenta

Prostaglandin E(2) 9-keto reductase (9-KPR) activity shifts reversibly PGE(2) into PGF(2 alpha) and may be responsible for the control of prostaglandins (PGs) levels in, among others, placental tissues. The retention of fetal membranes in cows is the postpartum disorder where the disturbances in PGs metabolism have been reported. It has been argued whether these disturbances are due to alterations in 9-KPR activity. In this study, the activity of the enzyme was determined in maternal and fetal bovine placental tissues which were divided into 6 groups as follows: (A) caesarian section before term without retained fetal membranes (n=10), (B) caesarian section before term with retained fetal membranes (n=10), (C) caesarian section at term without retained fetal membranes (n=12), (D) caesarian section at term with retained fetal membranes (n=12), (E) spontaneous delivery at term without retained fetal membranes (n=12), (F) spontaneous delivery at term with retained fetal membranes (n=12). The enzyme activity was measured spectrophotometrically and expressed in nanokatals (nkat) per protein content. The activity increased towards parturition and was significantly higher in maternal than in fetal part of placenta in all groups examined. The significantly higher values in retained than in not retained placental tissues were observed in the samples examined. The present results indicate that the disturbances in 9-KPR activity in bovine retained placenta exist but their reasons still require further experiments.     

The levels of lipid peroxidation products in bovine retained and not retained placenta

Unsaturated fatty acids undergo peroxidation processes. The presence of elevated levels of metabolites of lipid peroxidation processes may cause alterations in metabolic pathways which may lead to disturbances and clinical symptoms of illnesses. One such illness may be retention of fetal membranes (RFM) in cows. The aim of the study was the determination of thiobarbituric acid (TBA) reactive substances, conjugated dienes and hydroperoxides in bovine retained and not retained placenta in order to describe the oxidative status of cows affected with RFM. Placental samples were collected immediately after spontaneous delivery or caesarian section before term and at term and divided into 6 groups, with 16 animals in each group, as follows: A) caesarian section before term without RFM: B) caesarian section before term with RFM: C) caesarian section at term without RFM: D) caesarian section at term with RFM: E) spontaneous delivery at term without RFM; F) spontaneous delivery with RFM. The levels of TBA reactive substances, conjugated dienes and hydroperoxides were measured spectrophotometrically. The levels of all parameters were statistically significantly higher in the maternal than in the fetal part of placenta and the lowest in preterm groups. Statistically significantly higher concentrations in retained placenta samples than in control animals were observed. In conclusion, the metabolites of lipid peroxidation processes are elevated in cases of retained placenta and their presence might influence directly and/or indirectly the improper release of fetal membranes in cows.     

Determination of 8-iso-prostaglandin F(2alpha) in exhaled breath condensate using combination of immunoseparation and LC-ESI-MS/MS

Rapid and precise method for the determination of 8-iso-prostaglandin F(2alpha), an essential marker of the oxidative stress, in exhaled breath condensate (EBC) was developed. The protocol consisted of stable isotope dilution, immunoseparation combined with selective and sensitive LC-ESI-MS/MS operated in multiple reaction monitoring (MRM) mode. The imprecision of the developed method was below 8.8%, the parameter of mean inaccuracy was determined as <9.6% (0-250pg of 8-iso-prostaglandin F(2alpha)/ml EBC). The limit of detection (LOD) was 1 pg/ml EBC and limit of quantification (LOQ) 5 pg/ml EBC. A significant difference in 8-iso-prostaglandin F(2alpha) content between the group of asbestosis patients and healthy volunteers was found.     

F2-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome

Oxidative damage has been reported in Rett syndrome (RTT), a pervasive developmental disorder caused in up to 95% of cases by mutations in the X-linked methyl-CpG binding protein 2 gene. Herein, we have synthesized F(2)-dihomo-isoprostanes (F(2)-dihomo-IsoPs), peroxidation products from adrenic acid (22:4 n-6), a known component of myelin, and tested the potential value of F(2)-dihomo-IsoPs as a novel disease marker and its relationship with clinical presentation and disease progression. F(2)-dihomo-IsoPs were determined by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Newly synthesized F(2)-dihomo-IsoP isomers [ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP] were used as reference standards. The measured ions were the product ions at m/z 327 derived from the [M-181](-) precursor ions (m/z 597) produced from both the derivatized ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP. Average plasma F(2)-dihomo-IsoP levels in RTT were about one order of magnitude higher than those in healthy controls, being higher in typical RTT as compared with RTT variants, with a remarkable increase of about two orders of magnitude in patients at the earliest stage of the disease followed by a steady decrease during the natural clinical progression. hese data indicate for the first time that quantification of F(2)-dihomo-IsoPs in plasma represents an early marker of the disease and may provide a better understanding of the pathogenic mechanisms behind the neurological regression in patients with RTT.     

Transcardiac 8-iso-prostaglandin F(2 alpha)generation from acute myocardial infarction heart: insight into abrupt reperfusion and oxidant stress

8-iso-prostaglandin F(2 alpha)(8-iso-PGF(2 alpha)), a representative isoprostane, has been reported to be a reliable marker for oxidant stress in vivo. To examine if 8-iso-PGF(2 alpha)is generated in patients with acute myocardial infarction (AMI), we measured the level of immunoreactive 8-iso PGF(2 alpha)in the great cardiac vein as well as classical eicosanoids, 6-keto-prostaglandin F(1 alpha)(6-keto-PGF(1 alpha)) and thromboxane B(2)(TXB(2)) in the process of urgent coronary balloon angioplasty. Fourteen patients with anterior AMI were divided into two groups: the totally occluded (n=7) and the already perfused groups (n=7). In the former, transient elevation of 8-iso-PGF(2 alpha)was observed immediately after the angioplasty, i.e. the ratio of post-angioplasty level to pre-level was approximately 2.4 for 8-iso-PGF(2 alpha), 14 for 6-keto-PGF(1 alpha), and 5 for TXB(2). In the already perfused group, the levels of these eicosanoids were unchanged. In the totally occluded group, peak creatine phosphokinase in a peripheral vein was correlated with the level of 8-iso-PGF(2 alpha)(r(2)=0.841, P<0.01), but not with those of the other two eicosanoids. In conclusion, transcardiac 8-iso-PGF(2 alpha)generation is a reliable marker for the size of myocardium exposed to oxidant stress.     

Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes

We measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in human leukocytes from healthy donors to evaluate oxidative DNA damage and its correlation with smoking, physical exercise, and alcohol consumption. A significant increase in oxidative DNA damage was induced by cigarette smoke, with the mean level of 8-OHdG being significantly higher in smokers (33.1 +/- 10.6 per 10(6) 2-deoxyguanosine (dG) [mean +/- SE], n = 16) compared with nonsmokers (15.3 +/- 1.8 per 10(6) dG, n = 31) and former smokers (17.8 +/- 1.5 per 10(6) dG, n = 9). The highest values were observed after smoking more than 10 cigarettes per day (41.8 +/- 17.1 per 10(6) dG, n = 9). A large interindividual variation in 8-OHdG levels was observed in all analyzed groups. We also observed a correlation between 8-OHdG levels and age in nonsmokers and former smokers. Neither frequency of physical exercise nor alcohol drinking significantly modified 8-OHdG levels in leukocytes.     

Oxidative stress and 8-iso-prostaglandin F(2alpha) induce ectodomain shedding of CD163 and release of tumor necrosis factor-alpha from human monocytes

CD163 is a membrane glycoprotein of the cysteine-rich scavenger receptor superfamily. Upon an inflammatory stimulus CD163 undergoes ectodomain shedding and the soluble protein has been shown to play a role in downregulation of inflammation. The purpose of the present study was to identify a physiological activator of CD163 shedding that is consistently present under inflammatory conditions. Therefore, we elucidated whether oxidative stress or 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) is involved in shedding of CD163. Oxidative stress induced by H(2)O(2) or a NO donor as well as 8-iso-PGF(2alpha) induced significant shedding of CD163. In contrast, release of CD163 was not stimulated by PGF(2alpha). We identified both calcium and reactive oxygen species as common cellular mediators of CD163 release. Since shedding of both CD163 and tumor necrosis factor-alpha (TNFalpha) is known to be mediated by a TIMP-3-sensitive metalloproteinase we examined whether release of TNFalpha was induced by the same mediators that trigger shedding of CD163. Only oxidative stress generated by H(2)O(2) as well as 8-iso-PGF(2alpha) and PGF(2alpha) enhanced TNFalpha secretion. Thus, we identified novel common and divergent activators of shedding of CD163 and TNFalpha. These inducers of shedding are present in inflammation and might play an important role in membrane protein cleavage.     

Control of bovine uterine prostaglandin F2 alpha release in vitro

Prostaglandin F2 alpha (PGF2 alpha) release from the uterus causes luteolysis in ruminants, and oxytocin is thought to be a regulator of this release. In the present study, we have examined the mechanisms involved in oxytocin stimulation of PGF2 alpha secretion by bovine endometrium in vitro. Endometrial tissue explants, obtained from heifers at Day 19 or 20 (n = 3) and Day 0 (estrus, n = 5) of the estrous cycle, were incubated for 2 h and 6 h, and PGF2 alpha concentration in the medium was determined by radioimmunoassay (RIA). Basal PGF2 alpha release increased for up to 6 h and was significantly stimulated after 2 h of incubation with 100 microU and 1000 microU of oxytocin at Day 0 but not at Day 19 or 20. Secretion of PGF2 alpha was not affected by cholera toxin (10 ng/ml) or the cyclic nucleotide analogs dibutyryl cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic guanosine 3',5'-monophosphate at a concentration of 1 mM. A protein kinase A inhibitor (500 microM) had no effect on the oxytocin-induced release of PGF2 alpha. Both the phorbol ester, 12-myristate-13-acetate (100 mM), and the non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol (500 microM), significantly stimulated PGF2 alpha secretion to the same extent as oxytocin. Neither basal nor stimulated PGF2 alpha release was affected by the calcium ionophore A23187 (0.1-5.0 microM). However, PGF2 alpha secretion was sensitive to cycloheximide (1 microgram/ml) suggesting that protein synthesis may be involved. In conclusion, these data suggest that the stimulation of PGF2 alpha by oxytocin is via the protein kinase C effector pathway.     

Occurrence and properties of a prostaglandin F2alpha receptor in bovine corpora lutea.

Prostaglandin F2alpha was specifically bound by a particulate fraction from bovine corpora lutea. The rate constants for the association (7.5 X 10(3) M-1 S-1) and dissociation (2.1 X 10-4 S-1) reactions gave a dissociation constant of 2.8 X 10(-8) M which is similar to that determined from a Scatchard plot of binding data at equilibrium (5 X 10(-8) M). The receptor was stable for several hours at 23 degrees C but was rapidly destroyed at 37 degrees C. The pH optimum for the binding reaction was 6.3. The receptor had high specificity for prostaglandin F2alpha and had much lower affinities for other prostaglandins. Luteinizing and follicle-stimulating hormones had no effect on the prostaglandin F2alpha-receptor interaction.     

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.     

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a reliable oxidative stress marker in hypertension

The potential use of oxidative stress products as disease markers and progression is an important aspect of biomedical research. In the present study, the quantification of urine 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) concentration has been used to express the oxidation status of hypertensive subjects.

8-oxo-dG has been simultaneously isolated and assayed in nuclear (nDNA) and mitochondrial DNA (mtDNA). In addition, oxidative stress of mononuclear cells has been estimated by means of GSH and GSSG levels and GSSG/GSH ratio in hypertensive subjects before and after antihypertensive treatment. It is shown that oxidative stress decreases significantly in hypertensive patients after treatment the effect being accompanied by reduction of their blood pressure.

A significant correlation is observed comparing the yield of urine 8-oxo-dG and that isolated from mitochondria DNA. Moreover, urinary excretion of 8-oxo-dG also correlates with the GSSG/GSH ratio of cells. Conclusion: urine 8-oxo-dG assay is a good marker for monitoring oxidative stress changes in hypertensives.     

Localization of a prostaglandin F2alpha receptor in bovine Corpus luteum plasma membranes.

The distribution of a prostaglandin F2alpha receptor in various subcellular fractions from bovine corpora lutea obtained by differential and gradient centrifugation paralleled very closely the distribution in these fractions of 5'-nucleotidase, a marker enzyme for plasma membranes. The fractions most enriched in the receptor and 5'-nucleotidase were relatively free of mitochondria and lysosomes but were contaminated to some extent by elements of the endoplasmic reticulum. From these results it can be concluded that the prostaglandin F2alpha receptor is localized on the plasma membranes of the corpus luteum cells. A simple method is described for the purification of plasma membranes from bovine corpora lutea by differential centrifugation.