Urinary F2-isoprostanes Research Articles

No difference in plasma or urinary F2-isoprostanes among patients with Huntington's disease or Alzheimer's disease and controls

No difference in plasma or urinary F2-isoprostanes among patients with Huntington's disease or Alzheimer's disease and controls

A High Salt Diet Increases Renal Nitrotyrosine Formation and Urinary Excretion of F 2 -Isoprostanes and Protein in Dahl Salt-Sensitive Rats

P28 Superoxide formation causes tissue injury in many disease processes, and interaction between superoxide and nitric oxide forms peroxynitrite, a very strong oxidant. An index of peroxynitrite damage to tissues is nitrotyrosine formation. Also, F 2 -isoprostanes have been shown to be a reliable marker of oxidative stress in vivo. Recently, oxidative stress was shown to contribute to the maintenance of hypertension in the SHR; however, whether oxidative stress is involved in the renal damage that occurs in Dahl salt-sensitive (S) hypertension is not clear. Our goal was to determine the role of oxidative stress in the renal damage that occurs in Dahl S/Rapp rats on high Na intake by determining the urinary excretion of F 2 -isoprostanes and renal nitrotyrosine formation and determining urinary protein excretion as an index of renal damage. Studies were conducted in S rats on a low salt (0.3% NaCl) or a high salt (8% NaCl) diet for 3 weeks. After 3 weeks urine was collected continuously for 24 hours, and urinary F 2 -isoprostane concentration was measured with a gas chromatography/mass spectrometric assay. Renal nitrotyrosine formation was determined by immunohistochemistry and urinary protein excretion by the Bradford method. We found that high Na intake significantly increased urinary F 2 -isoprostane excretion to 24.2±1.7 ng/day compared with 9.7±0.8 ng/day in rats on a low Na diet, P<0.05. Renal glomerular nitrotyrosine was prevalent in the high Na group compared to the low Na group. Urinary protein excretion was 274±32 mg/day in high Na rats and 55±11 mg/day in low Na rats, P<0.05. The results suggest that oxidative stress is significantly increased in Dahl S rats on a high salt diet, which possibly contributes to the progression of renal damage.

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 μL) 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-PGF2α 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-PGF2α 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.

Measurement of Urinary F2-Isoprostanes as Markers of in Vivo Lipid Peroxidation—A Comparison of Enzyme Immunoassay with Gas Chromatography/Mass Spectrometry

This study aimed at comparing the two most commonly utilized methods for measuring urinary F2-isoprostanes, currently considered one of the best available markers of in vivo lipid peroxidation. The F2-isoprostanes were measured in 24-h urine samples from 14 male subjects using electron capture negative ionization gas chromatography–mass spectrometry (ECNI–GCMS) with 8-iso-PGF2α-d4 as an internal standard and compared with levels obtained using an enzyme immunoassay (EIA, 8-iso-PGF2α kit, Cayman Chemical Co.). The methods were compared using Pearson correlation coefficients, and Bland–Altman plots were constructed for the difference in F2-isoprostane against the average F2-isoprostane measured by either method. Weighted least-products regression was used to determine fixed bias (where there is a consistent difference between the methods) and proportional bias (where one method gives values higher or lower than the other method by an amount proportional to the size of the measurement). The correlation between F2-isoprostane levels obtained using EIA and GCMS methods, although significant, was poor (r = 0.628, P < 0.02). Comparison of the methods using the Bland–Altman analysis showed that there were wide limits of agreement between the two methods with only 28% of the values falling within the 95% confidence limits for the difference. The GCMS gave higher values with a mean difference of 298.1 pM (636.6, −40.2; P = 0.079), and a near significant linear association between the differences and the mean F2-isoprostane level (r = −0.559, P = 0.05). Weighted least-product regression analysis confirmed the presence of both significant fixed and proportional bias with the EIA giving lower levels of F2-isoprostanes at low concentrations and higher levels at higher concentrations. The cross-reactivity in the EIA of 8-iso-15(R)-PGF2α and 9β-PGF2α which coelute with the F2-isoprostane peak measured by GCMS was very low, 0.2 and 0.1%, respectively. The proportional bias observed between the methods may in part be due to differences in the relative amounts of 8-iso-15(R)-PGF2α, 9β-PGF2α, and 8-iso-PGF2α with increasing lipid peroxidation. This study shows that the measurements of F2-isoprostanes by EIA and GCMS are not equivalent. Therefore, comparison of levels derived using a GCMS method which estimates concentration from a peak encompassing a number of F2-isoprostane isomers, and levels derived from enzyme immunoassay measuring a specific isoprostane, may be inappropriate.

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 stressin vivo.Furthermore, 8-epi-PGF2α, 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 asin vivomarkers of oxidant stress in clinical and experimental settings.

Acute cholestasis-induced renal failure: Effects of antioxidants and ligands for the thromboxane A2 receptor

Acute biliary obstruction is associated with the development of renal impairment and oxidative stress. The F2-isoprostanes, formed during oxidant injury, are renal vasoconstrictors acting via thromboxane (TX)-like receptors. We determined whether the formation of F2-isoprostanes is increased in experimental cholestasis and whether thiol containing antioxidants or ligands for the TXA2 receptor could improve renal function. The effects on renal function of acute bile duct ligation (BDL) in the rat were studied for two days. The consequences of administration of N-acetylcysteine (NAC), alpha-lipoic acid (LA), the TX receptor antagonist (TXRA) BAYu3405, or placebo were then examined. BDL caused a reduction in creatinine clearance from 1.10 +/- 0.05 to 0.55 +/- 0.05 ml/min and sodium excretion from 52 +/- 3 to 17 +/- 3 micromol/hr. Urinary F2-isoprostanes increased from 14 +/- 2 to 197 +/- 22 pg/ml following BDL. Renal functional changes were ameliorated by NAC (creatinine clearance 0.73 +/- 0.05 ml/min), LA (0.64 +/- 0.03 ml/min), and a TXRA (0.90 +/- 0.15 ml/min); P < 0.05. Similarly, sodium excretion was increased from 17 +/- 3 micromol/hr (placebo) to 34 +/- 3 micromol/hr (NAC), 29 +/- 3 micromol/hr (LA), and 38 +/- 5 micromol/hr (TXRA); P < 0.005. Hepatic glutathione concentrations increased from 6.5 +/- 0.3 micromol/g (normal liver) to 8.8 +/- 0.5 micromol/g (NAC) and 7.7 +/- 0.3 micromol/g (LA), P < 0.01. However, only LA markedly inhibited F2-isoprostane formation (197 +/- 22 to 36 +/- 11 pg/ml creatinine clearance; P < 0.05). Urinary TXB2 excretion was elevated after BDL (2.2 +/- 0.5 to 111.1 +/- 20.3 pg/min) but was unaffected by NAC and LA. NAC, LA, and TXRA can partially prevent renal dysfunction in experimental cholestasis. The effects of the antioxidants are independent of their ability to inhibit lipid peroxidation or TX synthesis.

N-acetylcysteine prevents development of the hyperdynamic circulation in the portal hypertensive rat.

Partial portal vein ligation (PPVL) leads to the development of a hyperdynamic circulation. It is associated with elevated levels of tumor necrosis factor (TNF-alpha) and nitric oxide (NO) production, both of which can result in oxidant injury. In this study, we have investigated whether PPVL is associated with the development of oxidative stress, by measuring urinary F2-isoprostanes. In addition, we have examined whether N-acetylcysteine (NAC) can ameliorate oxidant injury and prevent the development of the hyperdynamic circulation. Urinary excretion of F2-isoprostanes increased sixfold following PPVL together with a significant increase in plasma nitrite and nitrate. Treatment with NAC inhibited the formation of F2-isoprostanes as well as the increase in plasma nitrite and nitrate. Hemodynamic studies in anesthetized rats showed that following PPVL, cardiac output and portal pressure increased, and systemic vascular resistance decreased, consistent with the development of a hyperdynamic circulation. These changes were prevented by chronic administration of NAC. We conclude that NAC prevents the development of the hyperdynamic circulation and that the formation of reactive oxygen species may be important in the pathogenesis of these hemodynamic changes.

Evolving lipoprotein risk factors: lipoprotein(a) and oxidized low-density lipoprotein

Cardiovascular disease is the leading cause of morbidity and mortality in Westernized populations. Evolving lipoprotein risk factors include LDL oxidation and lipoprotein(a) [lp(a)]. Several lines of evidence support a role for oxidatively modified LDL in atherogenesis and its in vivo existence. There are both direct and indirect measures of oxidative stress. The most relevant direct measure of lipid peroxidation is urinary F2 isoprostanes. The most common indirect measure of LDL oxidation is quantifying the lag phase of copper-catalyzed LDL oxidation by assaying conjugated diene formation. Lp(a) is increased in patients with cardiovascular and cerebrovascular disease. However, not all prospective studies have confirmed a positive relationship between Lp(a) and cardiovascular events. Lp(a) appears to present three major problems: standardization of the assay, establishing its role in atherogenesis, and the lack of an effective therapy that can substantially lower Lp(a) concentrations. Thus, at the present time, Lp(a) concentrations should not be recommended for the general population but be reserved for patients with coronary artery disease without established risk factors, young patients with coronary artery disease or cerebrovascular disease, or a family history of premature atherosclerosis and family members of an index patient with increased concentrations of Lp(a). Although both LDL oxidation and Lp(a) are evolving risk factors for cardiovascular disease, more data are needed before they become part of the established lipoprotein repertoire.

Increased formation of the isoprostanes IPF2alpha-I and 8-epi-prostaglandin F2alpha in acute coronary angioplasty: evidence for oxidant stress during coronary reperfusion in humans.

The role of oxidant stress in cardiac ischemia/reperfusion injury in humans remains controversial. This is due, in part, to the limitations of available indices of oxidant stress in vivo. Isoprostanes are stable, free radical-catalyzed products of arachidonic acid. We assessed their formation in patients undergoing coronary reperfusion via percutaneous transluminal coronary angioplasty (PTCA). We developed specific, mass spectrometry assays for two structurally distinct F2 isoprostanes, 8-epi-PGF2alpha and IPF2alpha-I. Urine samples for isoprostane determination were collected in patients undergoing coronary arteriography (n=11), elective PTCA (n=15), and angiography after thrombolysis for acute myocardial infarction (MI) (n=10). Urinary levels (pmol/mmol creatinine) of both isoprostanes were markedly increased from baseline in the first 6 hours after PTCA for acute MI (105+/-17.8 versus 230+/-66 for 8-epi-PGF2alpha [P=.009] and 466+/-91 versus 833+/-153 for IPF2alpha-I [P=.001]) and returned toward preprocedural values by 24 hours (122+/-18 for 8-epi-PGF2alpha and 457+/-102 for IPF2alpha-I). There was a slight increase in urinary 8-epi-PGF2alpha levels (64.7+/-9.5 versus 84.9+/-10.6; P=.02) after diagnostic coronary arteriography and elective PTCA (88.7+/-7.5 versus 114.3+/-16.1; P=.01). A striking correlation was observed (r=.68, P<.0001; n=33) between urinary 8-epi-PGF2alpha and IPF2alpha-I levels in patients receiving thrombolytic agents for acute MI. Urinary F2 isoprostane levels are elevated in patients after treatments resulting in reperfusion for acute MI. These findings provide evidence consistent with increased oxidant stress in vivo in this setting. Measurement of urinary isoprostanes may offer a noninvasive approach to the assessment of oxidant stress and the efficacy of antioxidant therapies in these syndromes.