William E. Zackert

Increased CSF F2-isoprostane concentration in probable AD

Objective: To quantify F2-isoprostane levels in CSF obtained from the lumbar cistern of patients with AD, ALS, and controls. Background: Studies of human postmortem tissue and experimental models have suggested a role for oxidative damage in the pathogenesis of several neurodegenerative diseases, especially AD and ALS. F2-isoprostanes are exclusive products of free-radical–mediated peroxidation of arachidonic acid that have been widely used as quantitative biomarkers of lipid peroxidation in vivo in humans. Recently, we showed that F2-isoprostane concentrations are significantly elevated in CSF obtained postmortem from the lateral ventricles of patients with definite AD compared with controls. Methods: F2-isoprostanes were quantified by gas chromatography/negative ion chemical ionization mass spectrometry. Results: CSF F2-isoprostanes were increased significantly in patients with probable AD, but not in ALS patients, compared with controls. Conclusions: Increased CSF F2-isoprostanes are not an inevitable consequence of neurodegeneration and suggest that increased brain oxidative damage may occur early in the course of AD.

IDENTIFICATION OF THE MAJOR URINARY METABOLITE OF THE F2-ISOPROSTANE 8-ISO-PROSTAGLANDIN F2ALPHA IN HUMANS

F2-isoprostanes are prostaglandin-like products of nonenzymatic lipid peroxidation. Measurement of levels of endogenous unmetabolized F2-isoprostanes has proven to be a valuable approach to assess oxidative stress in vivo. However, measurement of levels of urinary metabolites of F2-isoprostanes in timed urine collections offers an advantage over measuring unmetabolized F2-isoprostanes, e.g. in a plasma sample, in that it can provide an integrated index of isoprostane production over time. Therefore, we sought to identify the major urinary metabolite in humans of one of the more abundant F2-isoprostanes produced, 8-iso-prostaglandin F2α (8-iso-PGF2α). 20 μCi of tritiated 8-iso-PGF2α was infused over 1 h into a male volunteer. 75% of the infused radioactivity was excreted into the urine during the following 4.5 h and was combined with urine collected for 4 h from a rhesus monkey following infusion of 500 μg of unlabeled 8-iso-PGF2α. Urinary metabolites were isolated and purified by adsorption chromatography and high pressure liquid chromatography. The major urinary metabolite, representing 29% of the total extractable recovered radioactivity in the urine, was structurally identified by gas chromatography and mass spectrometry as 2,3-dinor-5,6-dihydro-8-iso-prostaglandin F2α. The identification of 2,3-dinor-5,6-dihydro-prostaglandin F2α as the major urinary metabolite of 8-iso-prostaglandin F2α provides the basis for the development of methods of assay for its quantification as a means to obtain an integrated assessment of oxidative stress status in humans.

THE ISOPROSTANES: UNIQUE PROSTAGLANDIN-LIKE PRODUCTS OF FREE-RADICAL-INITIATED LIPID PEROXIDATION*

The discovery of IsoPs as products of nonenzymatic lipid peroxidation has opened up new areas of investigation regarding the role of free radicals in human physiology and pathophysiology. The quantification of IsoPs as markers of oxidative stress status appears to be an important advance in our ability to explore the role of free radicals in the pathogenesis of human disease. An important need in the field of free-radical medicine is information regarding the clinical pharmacology of antioxidant agents. Because of the evidence implicating free radicals in the pathogenesis of a number of human diseases, large clinical trials are planned or underway to assess whether antioxidants can either prevent the development or ameliorate the pathology of certain human disorders. However, data regarding the most effective doses and combination of antioxidant agents to use in these clinical trials is lacking. As mentioned previously, administration of antioxidants suppresses the formation of IsoPs, even in normal individuals. Thus, measurement of IsoPs may provide a valuable approach to define the clinical pharmacology of antioxidants. In addition to being markers of oxidative stress, several IsoPs possess potent biological activity. The availability of additional IsoPs in synthetic form should broaden our knowledge concerning the role of these molecules as mediators of oxidant stress. Despite the fact that considerable information has been obtained since the initial report of the discovery of IsoPs [6], much remains to be understood about these molecules. With continued research in this area, we believe that much new information will emerge that will open up additional important new areas for future investigation.

The Magnitude of Brain Lipid Peroxidation Correlates with the Extent of Degeneration but Not with Density of Neuritic Plaques or Neurofibrillary Tangles or with APOE Genotype in Alzheimer's Disease Patients

Numerous post mortem studies have demonstrated increased accumulation of lipid peroxidation products in diseased regions of Alzheimers disease (AD) brain; however, few have used techniques that quantify the magnitude of lipid peroxidation in vivo. F(2)-isoprostanes (F(2)-IsoPs) are exclusive products of free radical-mediated peroxidation of arachidonic acid, and their quantification has been widely used as an in vivo biomarker of the magnitude of lipid peroxidation. We have determined F(2)-IsoP concentrations in lateral ventricular fluid (VF) from 23 AD and 12 age-matched controls and correlated these with neuropathological and genetic markers of AD. VF F(2)-IsoP levels were significantly elevated in AD patients compared with controls (p < 0.01) and were significantly correlated with three different measures of brain degeneration: reduction in brain weight (p < 0.01), degree of cortical atrophy (p < 0.01), and Braak stage (p = 0.02). When analysis was restricted to AD patients only, VF F(2)-IsoP levels still were significantly correlated to reduction in brain weight and degree of cortical atrophy (p < 0.05). VF F(2)-IsoP concentrations were not related to density of neuritic plaques or neurofibrillary tangles in seven brain regions, or to the number of epsilon4 alleles of the apolipoprotein E gene (APOE). These data suggest that the magnitude of brain lipid peroxidation is closely related to the extent of brain degeneration in AD but is not significantly influenced by the density of neuritic plaques or neurofibrillary tangles, or the number of epsilon4 alleles of APOE.

Cerebrospinal fluid F2-isoprostanes are elevated in Huntington’s disease

Numerous investigations using a variety of experimental model systems have suggested that oxidative stress in the context of impaired energy metabolism may be an important element in the pathogenesis of Huntington’s disease (HD).1 To test further the hypothesis that oxidative damage may contribute to the progression of HD, we measured a quantitative and specific marker of lipid peroxidation, F2-isoprostanes (F2-IsoP), in CSF from HD patients early in the course of disease. This was then compared with control subjects and patients with another degenerative disease of the basal ganglia that also has been associated with increased oxidative damage: multiple system atrophy—striatonigral degeneration type (MSA-P).2 Following informed consent, CSF was aspirated from the lumbar cistern, and 1 to 2 mL were immediately frozen at −80 °C and later used for F2-IsoP quantification as described.3 All CSF had protein and glucose concentrations as well as cell counts within normal ranges. Statistical comparisons were made with GraphPad Prism 2.0 software (San Diego, CA). Control subjects (n = 23) were hospitalized patients who underwent diagnostic evaluation …

Measurement of F2- isoprostanes and isofurans using gas chromatography-mass spectrometry.

F2-Isoprostanes (IsoPs) are isomers of prostaglandin F2α formed from the nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Since discovery of these molecules by Morrow and Roberts in 1990, F2-IsoPs have been shown to be excellent biomarkers as well as potent mediators of oxidative stress in vivo in humans. Isofurans (IsoFs) are also oxidation products generated from the nonenzymatic oxidation of arachidonic acid. IsoFs are preferentially formed instead of F2-IsoPs in settings of increased oxygen tension. The protocol presented herein is the current methodology that our laboratory uses to quantify F2-IsoPs and IsoFs in biological tissues and fluids using gas chromatography/mass spectrometry (GC/MS). A variety of analytical procedures to measure F2-IsoPs, including other GC/MS methods and liquid chromatography/MS and immunological approaches, are reported in the literature. This method provides a very low limit of quantitation and is suitable for analysis of both F2-IsoPs and IsoFs from a variety of biological sources including urine, plasma, tissues, cerebral spinal fluid, exhaled breath condensate, and amniotic fluid, among others.

Human colorectal cancer cells efficiently conjugate the cyclopentenone prostaglandin, prostaglandin J2, to glutathione

Cyclopentenone prostaglandins (PGs), particularly those of the J-series, affect proliferation and differentiation in a number of cell lines. J-ring PGs have been shown to be ligands for the peroxisome proliferator-activated receptor (PPAR)-gamma and to modulate NF-kappaB-mediated gene transcription. We have previously reported that large quantities of eicosanoids, including PGJ(2), are produced by the human colorectal cancer cell line HCA-7 while lesser amounts of Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) are formed. In this and other cell lines, cyclopentenone PGs have been shown to increase cell proliferation, but factors that influence their formation and metabolism are poorly understood. Unlike other PGs, cyclopentenone PGs contain alpha,beta-unsaturated carbonyl groups that readily adduct various biomolecules such as glutathione (GSH) in vitro. We now report that in HCA-7 cells, PGJ(2) is largely metabolized by conjugation to GSH. Characterization of the adducts by liquid chromatography (LC)-mass spectrometry (MS) revealed two major metabolites consisting of (1) a novel GSH conjugate in which the carbonyl at C-11 of PGJ(2) is reduced and (2) intact PGJ(2) conjugated to GSH. Approximately 70% of the PGJ(2) added to HCA-7 cells was esterifed to GSH after 2 h of incubation, suggesting this pathway represents the major route of metabolic disposition of PGJ(2) in HCA-7 cells.

Improved assay for the quantification of the major urinary metabolite of the isoprostane 15-F2t-isoprostane (8-iso-PGF2α) by a stable isotope dilution mass spectrometric assay

BACKGROUND The F(2)-isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical catalyzed peroxidation of arachidonic acid. One IsoP, 15-F(2t)-IsoP (8-iso-PGF(2alpha)), has been shown to be formed in abundance in vivo and to exert potent biological activity. METHODS As a means to assess the endogenous production of this compound, we previously developed a method to quantify the major urinary metabolite of 15-F(2t)-IsoP, 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (2,3-dinor-5,6-dihydro-8-iso-PGF(2alpha), 15-F(2t)-IsoP-M ), by gas chromotography (GC)/negative ion chemical ionization mass spectrometry (MS) employing stable isotope dilution methodology. While useful, we found that the assay occasionally suffered from the presence of impurities that co-elute on GC with 15-F(2t)-IsoP-M, making the measurement of this compound difficult. We now report a modified assay for the quantification of 15-F(2t)-IsoP-M employing GC/MS that alleviates this problem. RESULTS Precision of the assay is +/-7% and the accuracy is 96%. The lower limit of sensitivity is approximately 8 pg. Normal concentrations of this metabolite in urine were found to be 0.46+/-0.09 ng/mg creatinine (mean+/-1 S.D.) Urinary excretion of 15-F(2t)-IsoP-M is markedly altered in situations associated with increased or decreased oxidant stress in vivo. CONCLUSIONS This assay provided a sensitive and accurate method to assess endogenous IsoP generation and can be used to further explore the role of oxidant injury in human disease.

Quantification of the major urinary metabolite of prostaglandin D2 by a stable isotope dilution mass spectrometric assay

Prostaglandin D2 (PGD2) has been found to be an important pathophysiological mediator in a number of human disorders. Thus a means to assess the endogenous production of PGD2 is of considerable clinical value. To accomplish this goal, we developed a method for the quantification of the major urinary metabolite of PGD2, 9 alpha, 11 beta-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid, by gas chromatography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an 18O4-labeled derivative for use as an internal standard. Novel derivatization and purification procedures were incorporated in the assay taking advantage of the ability of the lower side chain of this molecule to undergo cyclization at acidic pH to form a hemiketal, gamma-lactone, and uncyclization with methoximation. Precision of the assay is +/- 7% and accuracy is 96%. The lower limit of sensitivity is approximately 50 pg. Normal levels for the urinary excretion of this metabolite in 18 normal adults was found to be 1.08 +/- 0.72 ng/mg creatinine (mean +/- 2SD). Substantial elevations in the urinary excretion of the metabolite were found in clinical situations in which prostaglandin D2 has been shown to be released in increased quantities. Thus, this assay provides a sensitive and accurate method to assess endogenous production of prostaglandin D2 as a means to explore the pathophysiological role of prostaglandin D2 in human disease.

EVIDENCE FOR THE FORMATION OF A NOVEL CYCLOPENTENONE ISOPROSTANE, 15-A2T-ISOPROSTANE (8-ISO-PROSTAGLANDIN A2) IN VIVO

A2/J2-Isoprostanes (IsoPs) are prostaglandin (PG) A2/J2-like compounds that are produced in vivo as dehydration products of D2/E2-IsoPs. One A2-IsoP that should be formed is 15-A2t-IsoP (8-iso-PGA2). Analogous to cyclopentenone PGs, 15-A2t-IsoP readily undergoes nucleophilic addition to various biomolecules suggesting the compound is capable of exerting potent bioactivity. However, proof that it is definitively formed in vivo is lacking. Evidence is now presented that 15-A2t-IsoP, in fact, is generated in vivo by demonstrating that an endogenous A2-IsoP with a retention time on capillary GC identical with that 15-A2t-IsoP co-chromatographs through four high resolving HPLC purification procedures with authentic radiolabeled 15-A2t-IsoP.