Analysis of monohydroxyeicosatetraenoic acids and F2-isoprostanes as markers of lipid peroxidation in rat brain mitochondria

We have introduced two specific techniques for the quantitative measurement of monohydroxyeicosatetraenoic acids (HETEs) and F2-isoprostanes by gas chromatography-mass spectrometry/negative ion chemical ionization (GC-MS/NICI) to study lipid peroxidation in isolated rat brain mitochondria by iron/ascorbate. The analysis of HETEs involved hydrogenation, solid phase extraction on a C18-cartridge, formation of pentafluorobenzyl bromide and trimethylsilyl ether derivatives. In the case of F2-isoprostanes, the analytical procedure was similar to that of HETEs except that the hydrogenation step was omitted. We found that HETE content (sum of 5-, 8-12-, and 15-isomers) in freshly prepared rat brain mitochondria was 220 +/- 40pmol/mg protein. The corresponding content for the F2-isoprostane, 8-iso-PGF2alpha, was 0.21 +/-+/- 0.10 pmol/mg protein. HETEs and 8-iso-PGF2alpha were predominantly present in the esterified form. The content of both HETEs and 8-iso-PGF2alpha were increased in presence of iron/ascorbate as oxidation system. After 30 min incubation with Fe2+ ascorbate, the content of HETE isomers was increased about 6-fold compared with baseline levels whereas that for 8-iso-PGF2alpha was elevated 100-fold. Formation of HETEs and F2-isoprostanes corresponded to the consumption of arachidonic acid (AA) and alpha-tocopherol, respectively. There were almost no changes in the content of free (non-esterified) HETEs and 8-iso-PGF2alpha during the course of iron/ascorbate induced oxidation of the brain mitochondria. Our data provide the first direct evidence for the presence of HETEs and F2-isoprostanes in freshly isolated rat brain mitochondria and that esterified HETEs and 8-iso-PGF2alpha are predominantly generated during iron/ascorbate induced lipid peroxidation. Sensitive quantification of these products of non-enzymatic lipid peroxidation as indicators of oxidant injury opens new areas of investigation regarding the role of free radicals in the pathogenesis of human diseases. In addition, HETEs and F2-isoprostanes may be important mediators for mitochondrial functions.     

Quantification of 8-iso-prostaglandin-F(2alpha) and 2,3-dinor-8-iso-prostaglandin-F(2alpha) in human urine using liquid chromatography-tandem mass spectrometry

Quantification of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) has been suggested to be a reliable indicator of lipid peroxidation that may be related to in vivo free radical generation, oxidative damage, and antioxidant deficiency. We have developed a LC-MS/MS method to quantify 8-iso- PGF(2alpha) and its dinor metabolite, 2,3-dinor-8-iso-prostaglandin F(2alpha) (2,3-dinor-8-iso-PGF(2alpha)), in human urine samples. After an initial purification step using an automated C18 solid phase extraction procedure, the urine sample was injected directly into a liquid chromatography (LC) system and detected with tandem mass spectrometry. The detection limit of the assay was 9 pg for 8-iso-PGF(2alpha) and 3 pg for 2,3-dinor-8-iso-PGF(2alpha) with both inter- and intraday variations of less than 12%. The inaccuracies were less than 3% for both analytes at three different levels. The urinary excretion rate of 2,3-dinor-8-iso-PGF(2alpha) was higher than that of 8-iso-PGF(2alpha), and changed in proportion to the parent compound (R = 0.70, n = 60). Values obtained with this method showed good linear correlation to duplicate 8-iso-PGF(2alpha) measurements performed with GCMS (R = 0.97, n = 15). The mean excretion rates of 8-iso-PGF(2alpha) and 2,3-dinor-8-iso-PGF(2alpha) were significantly higher in smokers than in nonsmokers (0.53 +/- 0.37 vs. 0.25 +/- 0.15 microg/g creatinine, p = 0.002 for 8-iso-PGF(2alpha) and 8.9 +/- 3.8 vs. 4.6 +/- 2.6 microg/g creatinine, p = 0.003 for 2,3-dinor-8-iso-PGF(2alpha), respectively). The excellent accuracy, reproducibility, and high throughput of this method should permit it to be used in large clinical studies and standard clinical laboratories.     

Time course and attenuation of ischaemia-reperfusion induced oxidative injury by propofol in human renal transplantation

Ischaemia-reperfusion injury resulting from interruption and restoration of blood flow might be related to free radical mediated oxidative stress and inflammation, and subsequently to post-surgery related complications. We studied the impact of renal transplantation on oxidative stress and inflammation by measuring F(2)-isoprostanes and prostaglandin F(2alpha), respectively, during transplantation and post-surgery. Additionally, due to earlier observations, two dissimilar anaesthetic agents (thiopentone and propofol) were compared to determine their antioxidative capacity rather than their anaesthetic properties. Blood samples were collected before, post-intubation, immediately, 30, 60,120, 240 min, and 12 and 24 h after reperfusion. Oxidative stress and inflammatory response were detected by measuring 8-iso-PGF(2alpha) (a major F(2)-isoprostane and a biomarker of oxidative stress) and 15-keto-dihydro-PGF(2alpha) (a major metabolite of PGF(2alpha) and a biomarker of COX-mediated inflammatory response), respectively. Reperfusion of the transplanted graft significantly increased plasma levels of 8-iso-PGF(2alpha). PGF(2alpha) metabolite levels, although elevated, did not reach statistical significance. In addition, significantly lower levels of 8-iso-PGF(2a) were observed in the propofol group compared to the thiopentone group. Together, these findings underline an augmented oxidative stress activity following an inflammatory response after human renal transplantation. Furthermore, propofol a well-known anaesthetic, counteracted oxidative stress by lowering the formation of a major F(2)-isoprostane.     

F2-isoprostane induced prostaglandin formation in the rabbit

F(2)-isoprostanes, non-enzymatic free radical mediated products of arachidonic acid, have shown to form during various oxidant stress status and have potent biological effects. This study investigates to what extent 8-iso-PGF(2alpha) (a major F(2)-isoprostane), a bioactive product of lipid peroxidation can modify endogenous prostaglandin F(2alpha) (PGF(2alpha)) formation since prostaglandins are inflammatory as well as potent vasoregulatory substances that modulate diverse important physiological functions, and also form during acute and chronic inflammation. An immediate appearance and disappearance of 8-iso-PGF(2alpha) was seen in both plasma and urine within a short interval after i.v. administration of 43 microg/kg of 8-iso-PGF(2alpha) to the rabbits. A successive but differential formation of PGF(2alpha) resulted in a rapid and pulsatile increase of plasma 15-keto-dihydro-PGF(2alpha), a major metabolite of primary PGF(2alpha). Later, this compound was excreted efficiently as intact compound into the urine during the 3 h of experiment. A 8-fold increase of PGF(2alpha) metabolite in plasma at 10 min and 12-fold increase in the urine at 30-60 after the i.v. administration of 8-iso-PGF(2alpha) was observed which continued throughout the 3 h of experiment. This observation suggests that pharmacologically administered or endogenously produced 8-iso-PGF(2alpha) during oxidant stress induces prostaglandin formation presumably through the classical cyclooxygenase-catalysed arachidonic acid oxidation which might be inflammatory itself to the cells and exerts further vasoconstrictive effects.     

Streamlined F2-isoprostane analysis in plasma and urine with high-performance liquid chromatography and gas chromatography/mass spectroscopy

F2-Isoprostanes in plasma and urine are generally determined by labor-intensive methods requiring sample purification by solid-phase extraction and thin-layer chromatography (TLC). A streamlined and more sensitive method for the measurement of esterified plasma F2-isoprostanes was developed by replacing these steps with high-performance liquid chromatography (HPLC) using an amino column with a hexane/2-propanol gradient. Pentafluorobenzyl esters of F2-isoprostanes were prepared and purified by HPLC, silylated, and then analyzed by gas chromatography (GC) with negative chemical ionization mass spectroscopy (NCI-MS). This method permits analysis with lower plasma volumes (100 microL) and greater sensitivity (to 10 pg; allowing detection to 50 pg/mL) than provided by other methods. Urinary F2-isoprostanes can also be efficiently quantified by this method, with 8-iso-PGF2alpha being identified as a major isomer. With this procedure, esterified plasma F2-isoprostanes were found to be 8.3-fold higher in an end-stage renal failure patient on hemodialysis and urinary 8-iso-PGF2alpha was 7.1-fold higher in a cigarette smoker than respective control subjects. This method, particularly the substitution of the TLC step common to other methods with HPLC, results in a more sensitive and reproducible assay.     

Benefits of prolonged gradient separation for high-performance liquid chromatography-tandem mass spectrometry quantitation of plasma total 15-series F-isoprostanes

The F(2)-isoprostanes are products of free-radical-induced oxidation of arachidonic acid (AA) that are stereoisomers of prostaglandin F(2alpha) (PGF(2alpha)). We describe a method for quantitation of several 15-series PGF isomers (15-PGFs) and AA by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS). Plasma samples were subjected to alkaline hydrolysis and acidified, and total (free + esterified) 15-PGFs and AA were extracted with organic solvents. The analytes were separated by gradient reverse-phase HPLC and detected by multiple reaction monitoring on a triple-quadrupole mass spectrometer, using deuterated internal standards for quantitation. The assay had a linear range of 1-40 pg of 8-iso-PGF(2alpha) on column and can quantify as little as 40 pg/mL (0.11 nM) in plasma. Outcomes significantly correlated (p < 0.0001) with data obtained by gas chromatography-mass spectrometry GC-MS or enzyme-linked immunosorbent assay. All plasma 15-PGF isomers increased over time with in vitro cigarette smoke exposure and correlated (p < 0.0001) with each other. The same strong inter-15-PGF correlations were observed in plasma from healthy young adult subjects. The coefficients of variation of HPLC-MS-MS measurements (24-32%) were smaller than those obtained by GC-MS (53%). Thus, HPLC-MS-MS potentially offers greater precision and allows quantitation of more compounds with simpler sample preparation than existing methods. Ours is the first validated quantitative assay using HPLC-tandem MS applied to plasma total 15-PGFs.     

Postocclusive reactive hyperemia inversely correlates with urinary 15-F2t-isoprostane levels in systemic sclerosis

Microvascular dysfunction and increased oxidative stress are major hallmarks of the systemic sclerosis disease process. The primary objective of this study was to test whether there is a link between peak postocclusive hyperemia and urinary levels of the F2-isoprostane 15-F2t-IsoP (8-iso-PGF2alpha) in patients suffering from systemic sclerosis. We enrolled 43 patients suffering from systemic sclerosis, 33 patients with primary Raynaud's phenomenon (RP), and 25 healthy volunteers. Microvascular function was assessed using the postocclusive hyperemia monitored by laser Doppler flowmetry. Endothelium-independent response was monitored after 0.4 mg sublingual nitroglycerin. Oxidative stress status was assessed by urinary levels of the F2-isoprostane 15-F2t-IsoP using GC-MS. The peak postocclusive vascular conductance was altered in subjects with systemic sclerosis and primary RP compared to controls (respectively 28 (7-48), 30 (13-48), and 39.9 (13-63) mV/mm Hg, p = 0.01). F2-isoprostanes were increased in the systemic sclerosis group compared to primary Raynaud's phenomenon and healthy controls (respectively 230 (155-387), 182 (101-284), and 207 (109-291) pg/mg, p = 0.006). In patients suffering from systemic sclerosis, there was a significant inverse correlation between F2-isoprostanes and postocclusive hyperemia, expressed as raw data (R = -0.45, p = 0.007) or as an increase over baseline (R = -0.28, p = 0.04). Conversely, no correlation was found with the nitroglycerin response. In conclusion, we provide evidence that there is an inverse correlation between postocclusive hyperemia and urinary F2-isoprostane levels in patients suffering from systemic sclerosis. Whether oxygen free radicals initiate the vascular dysfunction or whether there is an initial trigger that initiates both processes will need to be further clarified in future studies.     

In vivo effect of 8-epi-PGF2alpha on retinal circulation in diabetic and non-diabetic rats

Retinal hemodynamic responses to a F2-isoprostane, 8-epi-PGF2alpha, were quantitated in vivo in non-diabetic and diabetic rats using a video fluorescein angiography system. Vascular diameters and retinal mean circulation time were determined before and after 5 microl intra-vitreous injection of 8-epi-PGF2alpha (10(-5) to 10(-3) M), 10(-4) M 8-epi-PGF2alpha, + 10(-3) M SQ29,548 or 10(-3) M LCB2853 (two inhibitors of TXA2 receptor), 10(4) M 9beta-PGF2alpha, or the carrier in non-diabetic animals. Diabetic rats received either 8-epi-PGF2alpha 10(-4) M, or the carrier. Compared to control animals, diabetic rats presented in the basal state a venous vasodilation (P<0.01), without modification of retinal mean circulation time or blood flow. After intravitreous injection of 8-epi-PGF2alpha, a significant arterial vasoconstriction was observed in control but not in diabetic animals. This vasoconstriction was concomitant with increased retinal mean circulation time in control but not in diabetic rats, inducing an impaired reduction of blood flow. No vasoconstriction was observed after injection of either the carrier, 9beta-PGF2alpha or the isoprostane associated to the inhibitors of TXA2 receptors. This is the first direct observation that the isoprostane 8-iso-PGF2alpha is a potent vasoconstricting agent in the retina. It occurs at the arterial but not venous level, and is likely mediated through a TXA2-like receptor. Differences observed between control and diabetic animals suggest altered adaptative mechanisms toward vasoconstrictor substances (such as isoprostanes) in diabetic rats.     

The response of muscle interstitial F2-isoprostane (8-ISO-PGF2alpha) during dynamic muscle contractions in humans

8-Iso-prostaglandin F2alpha (8-iso-PGF2alpha) is a characteristic F2-isoprostane which is produced in humans via a free radical-catalysed lipid peroxidation mechanism of arachidonic acid, independent of the cycloxygenase pathway. The measurement of the plasma levels of 8-iso-PGF2alpha was shown to be the most reliable biochemical index of oxidant stress status in the human body. However, there is no reference in literature of local muscle interstitial 8-iso-PGF2alpha production during dynamic muscle contractions. The aim of the present study was to evaluate the response of 8-iso-PGF2alpha during intensive exercise with a cycle ergometer. Two microdialysis probes with CMA-60 microdialysis catheters were inserted into the vastus lateralis muscle of the right leg of six healthy male volunteers. After insertion, these microdialysis probes were attached to a perfusion pump that perfused ringer acetate solution at a rate of 0.3 microl/min. The dialysate fluid samples were collected: (a) during a 30 min rest period and (b) during a 30 min period of dynamic exercise with a cycle ergometer at 150 Watts. Our measurements showed that the levels of 8-iso-PGF2alpha in the interstitial fluid (IF) of the vastus lateralis muscle increase significantly during exercise (from 113.5 +/- 30.2 to 329.9 +/- 69.8 pg/ml, P = 0.05). In conclusion, dynamic muscle exercise produces a local increase of the IF levels of 8-iso-PGF2alpha due to local peroxidation injury of the contractive muscle. The microdialysis method is widely applied, easily repeated and it could contribute in evaluating the local lipid muscle peroxidation during intensive exercise.     

Estradiol reduces F2alpha-isoprostane production in cultured human endothelial cells

Free radical-generated F(2alpha)-isoprostanes are a group of compounds with vasoconstrictor properties. To investigate whether estradiol exerts antioxidant actions modifying F(2alpha)-isoprostane production, cultured human umbilical vein endothelial cells were exposed to estradiol and other compounds and F(2alpha)-isoprostanes were measured in culture medium. Exposure to 1 and 10 nM estradiol for 24 h reduced F(2alpha)-isoprostane production by 36 and 49%, respectively (P < 0.001 vs. control). Exposure to antiestrogens alone (ICI-182780 or EM-652) slightly reduced F(2alpha)-isoprostanes (P < 0.05 vs. control), but much less than exposure to estradiol (P < 0.05). ICI-182780 reversed the estradiol-induced reduction of F(2alpha)-isoprostane concentration (P < 0.05). Along with time-course analysis, these results suggest that estradiol effects were mediated through estrogen receptor-dependent and -independent mechanisms. Progestogens alone (progesterone or medroxyprogesterone acetate) did not modify F(2alpha)-isoprostane production at any of the tested concentrations (1, 10, and 100 nM). Progesterone completely reversed estradiol-induced reduction of F(2alpha)-isoprostane production (P < 0.05 vs. control and estradiol), but medroxyprogesterone acetate did not (P < 0.05 vs. control).     

An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry

We have developed an improved method for the measurement of F2-isoprostanes using stable isotope dilution capillary gas chromatography/electron capture negative ionization mass spectrometry (GC-ECNI-MS). The F2-isoprostane family consists of a series of chemically stable prostaglandin F2 (PGF2)-like compounds generated during peroxidation of arachidonic acid in phospholipids. There is evidence that measurement of F2-isoprostanes represents a reliable and useful index of lipid peroxidation and oxidant stress in vivo. Furthermore, 8-epi-PGF2alpha, which is one of the more abundant F2-isoprostanes, is biologically active, being a potent mitogen and vasoconstrictor of rat and rabbit lung and kidney, as well as a partial agonist of platelet aggregation. Measurement of F2-isoprostanes in biological samples is complex and has involved methods which utilize multiple chromatographic steps, including separation by thin-layer chromatography, leading to poor sample recovery. We now present an improved method for the measurement of plasma and urinary F2-isoprostanes using a combination of silica and reverse-phase extraction cartridges, high-performance liquid chromatography (HPLC), and GC-ECNI-MS. Different approaches to the derivatization of the F2-isoprostanes prior to GC-ECNI-MS are also addressed. The overall recovery of F2-isoprostanes is improved (approx 70% for urine) and the within and between assay reproducibility is 6.7% (n = 23) and 3.7% (n = 3), respectively. The mean urinary excretion of F2-isoprostanes in eight healthy males was 365 +/- 5 pmol/mmol creatinine and in three smokers 981 +/- 138 pmol/mmol creatinine. The mean total (free + esterified) plasma F2-isoprostane concentration was 952 +/- 38 pmol/liter, with a within and between assay reproducibility of 8% (n = 13) and 5.6% (n = 3), respectively. This improved method for the measurement of F2-isoprostanes represents a significant advance in terms of the rapidity and yield in the purification of biological samples. The inclusion of HPLC separation enables improved analysis of F2-isoprostanes by GC-MS. This methodology will assist in defining the role of F2-isoprostanes as in vivo markers of oxidant stress in clinical and experimental settings.     

Divergence in urinary 8-iso-PGF(2alpha) (iPF(2alpha)-III, 15-F(2t)-IsoP) levels from gas chromatography-tandem mass spectrometry quantification after thin-layer chromatography and immunoaffinity column chromatography reveals heterogeneity of 8-iso-PGF(2alpha). Possible methodological,mechanistic and clinical implications

Free radical-catalysed oxidation of arachidonic acid esterified to lipids leads to the formation of the F(2)-isoprostane family which may theoretically comprise up to 64 isomers. We have previously shown that the combination of TLC and GC-tandem MS (referred to as method A) allows for the accurate and highly specific quantification of 8-iso-PGF(2alpha) (iPF(2alpha)-III, 15-F(2t)-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF(2alpha) (i.e. 15(S)-8-iso-PGF(2alpha)) has been shown by others to be highly selective and specific for this 8-iso-PGF(2alpha) isomer when quantified by GC-MS. In the present study we established IAC for urinary 8-iso-PGF(2alpha) for subsequent quantification by GC-tandem MS (referred to as method B). This method was fully validated and found to be highly accurate and precise for urinary 15(S)-8-iso-PGF(2alpha). 8-iso-PGF(2alpha) was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF(2alpha) was determined to be 291+/-102 pg/mg creatinine by method A and 141+/-41 pg/mg creatinine by method B. Analysis of the combined through and wash phases of the IAC step, i.e. of the unretained compounds, by method A showed the presence of non-immunoreactive 8-iso-PGF(2alpha) at 128+/-55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF(2alpha) is heterogenous, with 15(S)-8-iso-PGF(2alpha) contributing by approximately 50%. PGF(2alpha) and other 8-iso-PGF(2alpha) isomers including 15(R)-8-iso-PGF(2alpha) are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF(2alpha). We assume that ent-15(S)-8-iso-PGF(2alpha) is also contributing by approximately 50% to urinary 8-iso-PGF(2alpha). This finding may have methodological, mechanistic and clinical implications.     

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.     

Association between oxidative stress and bone mineral density

Free radicals have been shown to be involved in bone resorption in vitro and in rodents. We studied the effect of oxidative stress on bone mineral density (BMD) in 48 women and 53 men from a population-based study. The levels of 8-iso-PGF(2alpha) (a major F(2)-isoprostane and a biomarker of oxidative stress) and a control, 15-keto-dihydro-PGF(2alpha) (a biomarker of inflammatory response), were measured in urinary samples and their association with BMD and quantitative ultrasound (QUS) measurements were examined. In multivariate linear regression analyses, 8-iso-PGF(2alpha) levels were negatively associated with both BMD and QUS. In contrast, no association was found for 15-keto-dihydro-PGF(2alpha). Our findings establish a biochemical link between increased oxidative stress and reduced bone density and provide a rational for further studies investigating the role of pro- and antioxidants in osteoporosis.     

Radioimmunological measurement of F(2)-isoprostanes after hydrolysis of lipids in tissues

8-Iso-prostaglandin F(2 alpha)(8-iso-PGF(2 alpha)) is a major isoprostane formed in vivo mainly by non-enzymatic peroxidation of arachidonic acid and a potential biomarker of oxidative injury. We have recently reported development of a specific radioimmunoassay for the measurement of free 8-iso-PGF(2 alpha)in plasma and urine. The aim of this study was to employ this radioimmunoassay to analyze the total amount of 8-iso-PGF(2 alpha)(sum of free and esterified) in liver tissues by using alkaline hydrolysis, and to apply it in an experimental model of carbon tetrachloride-induced lipid peroxidation in rats. Basal levels of total 8-iso-PGF(2 alpha)in hydrolyzed liver tissues of control rats were 6.5 times higher than the levels of free 8-iso-PGF(2 alpha). At maximum formation of total 8-iso-PGF(2 alpha)in the livers of carbon tetrachloride-treated rats, total levels of 8-iso-PGF(2 alpha)were almost 13 times higher than the levels of free 8-iso-PGF(2 alpha). In conclusion, high levels of 8-iso-PGF(2 alpha)in tissues can be quantified after hydrolysis both at basal conditions and in a model of increased lipid peroxidation. The methodology for measurement of total levels of 8-iso-PGF(2 alpha)in tissues may be suitable for future investigations of the location of oxidative injury in the body.     

Urinary excretion of 8-iso-PGF(2 alpha) in three patients during sepsis,recovery and state of health

Sepsis is known to be associated with oxidative stress. Novel markers of oxidative stress are now believed to be F2-isoprostanes which are produced in situ in phospholipids and subsequently released into circulation and excreted in the urine. This study, therefore, sought to investigate whether the excretion of the isoprostane, 8-iso-PGF(2 alpha), is elevated during sepsis. The excretion of 8-iso-PGF(2 alpha), in the 24 h urine of three patients was studied in the septic stage, during mobilisation and in the state of health by a radioimmunological method. Extrapolating the urinary excretion of 8-iso-PGF(2 alpha) over time showed an insignificant variation in the excretion values during 24 h. The amount of mean 24 h urinary 8-iso-PGF(2 alpha) was about similar in the septic stage and in the state of health but increased remarkably during mobilisation in two of the patients. We suggest that mobilisation of septic patients can be associated with an increase of oxidative stress which may stem from an increase in oxygen consumption and/or from a depletion of antioxidants leading to the enhanced formation of free radicals.     

Cardiopulmonary bypass as a cause of free radical-induced oxidative stress and enhanced blood-borne isoprostanes in humans

Free radicals are believed to be involved in postsurgery-related complications. We studied whether cardiopulmonary bypass (CPB) operation has any immediate impact on the initiation of oxidative stress and inflammatory response by measuring isoprostanes and prostaglandin F2alpha during and 24 h following CPB. The levels of 8-iso-PGF2alpha (a major F2-isoprostane and biomarker of oxidative stress) and 15-keto-dihydro-PGF2alpha (a major metabolite of PGF2alpha and biomarker of inflammatory response) were measured in frequently collected plasma samples before, during, and up to 24 h postsurgery in 21 patients. 8-Iso-PGF2alpha levels significantly increased within 3 min (p <.0001) and continued until 50 min (p <.0001) during CPB. On the contrary, no significant increase of inflammatory response indicator, 15-keto-dihydro-PGF2alpha was found during and up to 24 h postoperatively. These findings establish an increased free radical-induced oxidative stress activity rather than inflammatory response after CPB.     

Lipid peroxidation is increased in paraoxonase L55 homozygotes compared with M-allele carriers

Human serum paraoxonase (PON) is an antioxidative enzyme, which circulates on high-density lipoproteins and appears to use oxidized phospholipids as physiological substrates. PON M/L55 substitution changes the ability of PON to prevent lipid oxidation. Urinary 8-iso-PGF(2alpha) (one of F2 -isoprostanes) may represent a non-invasive in vivo index of free radical generation and we propose that PON might influence the biosynthesis of 8-iso-PGF(2alpha) in the vasculature. We studied the urinary excretion of 8-iso-PGF(2alpha) and related it to PON M/L55 genotypes in patients with type 2 diabetes mellitus (n = 55) and non-diabetic control subjects (n = 55). Urinary 8-iso-PGF(2alpha) was determined by competitive ELISA and the PON genotype by a PCR based restriction enzyme digestion method. LL homozygotes were compared to M-allele carriers (ML heterozygotes and MM homozygotes). The urinary excretion of 8-iso-PGF(2alpha) among non-diabetic non-smoking LL homozygotes was 3995.5 +/- 3352.8 ng/24-hour and among M-allele carriers 1689.8 +/- 1051.3 ng/24-hour (p = 0.017, ANCOVA; gender, hypertension, total cholesterol, triglycerides and LDL cholesterol as covariates). The excretion of 8-iso-PGF(2alpha), was increased in type 2 diabetes mellitus compared to non-diabetic control subjects. PON may thus protect against oxidative stress by destroying some biologically active lipids. Excretion of 8-iso-PGF(2alpha) is increased in type 2 diabetes, which may reflect oxidant injury.     

Relationship between levels of oxidative DNA damage, lipid peroxidation and mitochondrial membrane potential in young and old F344 rats

The extent of in vivo oxidative damage has been known to be cumulative over the period of the life of mammals. Our hypothesis is that there should be a positive correlation between the levels of 8-hydroxy-2'-deoxyguanosine (8OHdG) and 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) in major rat tissues. We also investigated whether increased level of oxidative stress causes a decrease in the mitochondrial membrane potential of peripheral lymphocytes of old rats using the MitoTracker Red fluorochrome. Our results show positive correlations between 8OHdG and 8-iso-PGF(2alpha) for liver, brain and kidney measured by HPLC-UV-ECD (electrochemical detector) and EIA methods, respectively. However, heart tissues show a negative correlation. The mitochondrial membrane potential of old rat lymphocytes records significant decrease compared with the young lymphocytes. Based on our results, we conclude that in ageing studies, specific tissues need to be examined in order to measure the localised DNA damage and lipid peroxidation as different tissues display different extent of oxidative damage. We believe this approach of using combined markers is useful to verify the true efficacy of health intervention studies in animals and humans. In addition, the isoprostane assay can be further developed looking at lipid peroxidation as a potential marker in ageing studies.     

Analyses of prostaglandin D2 metabolites in urine: comparison between enzyme immunoassay and negative ion chemical ionisation gas chromatography-mass spectrometry.

Measurements of the prostaglandin (PGD2) metabolite 9 alpha, 11 beta-PGF2 in unextracted urine performed by enzyme immunoassay (EIA) were compared with values obtained by negative chemical ionisation gas chromatography-mass spectrometry (NCI GC-MS). Values determined by NCI GC-MS were in the same range but consistently lower than those obtained by EIA, suggesting that other endogenous compounds could be contributing to the immunoreactivity. Isoprostanes were generated by autoxidation of arachidonic acid and the 9 alpha, 11 beta-PGF2 antibody demonstrated less than 0.7% crossreactivity to the mix, making it unlikely that isoprostanes in urine interfere with quantification of 9 alpha, 11 beta-PGF2 by EIA. This was further supported by the 70% reduction in immunoreactive material measured in urine after three days treatment in a healthy volunteer with the cyclooxygenase inhibitor ibuprofen. Purification of urine samples by reverse phase high-performance liquid chromatography (HPLC) revealed the presence of two immunoreactive compounds in addition to 9 alpha, 11 beta-PGF2. The compounds were identified as dinor compounds by NCI GC-MS. One of the compounds was identical to 9 alpha, 11 beta-2,3-dinor-PGF2 which was generated by beta-oxidation of 9 alpha, 11 beta-PGF2 and identified by electron impact (EI)-GC-MS. In conclusion, urinary 9 alpha, 11 beta-PGF2 concentrations measured by EIA represent the sum of 9 alpha, 11 beta-PGF2 and two isomers of its dinor metabolite. Thus, the direct EIA is fast, sensitive and sufficiently specific to monitor activation of the PGD2 pathway, thereby providing a valuable clinical tool to assess the status of mast cell activation in vivo.