News & Views !""# Oxidative Stress. Denis M- Callewaert President$ Oxford Biomedical Research$ Inc- Professor of Chemistry Oakland University

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Prostaglandin ELISAs Cycooxygenases Lipoxygenases Histamine ELISA Vascular Biology Plasminogen Activators tpa and urokinase PAI'* Fibronectin Vitronectin Signal Transduction Nitric Oxide Synthase

1 News & Views!""# Oxidative Stress Biomarker Assays Isoprostanes Lipid Peroxidation Myeloperoxidase Antioxidant Assays Total Antioxidants Glutathione Superoxide Dismutase Inflammation Eicosanoid Metabolism Prostaglandin ELISAs Cycooxygenases Lipoxygenases Histamine ELISA Vascular Biology Plasminogen Activators tpa and urokinase PAI'* Fibronectin Vitronectin Signal Transduction Nitric Oxide Synthase Ultrasensitive assays Antibodies Cyclic Nucleotide ELISAs Kinases & Phosphatases Recombinant proteins Antibodies Cancer Biology Oncogene products Cytokines Matrix Metalloproteases Xenobiotic Metabolism Cytochrome P&."s Glutathione Transferases Epoxide Hydrolase Proteomics: Immunodepletion of High Abundance Proteins Human ProteoMine Rodent ProteoMine Isolation of Modi(ed Proteins ToxPro(ler Denis M- Callewaert President$ Oxford Biomedical Research$ Inc- Professor of Chemistry Oakland University soprostanes$ a group of %& prostaglandin'like compounds$ are derived primarily by free radical' mediated peroxidation of free or esteri(ed arachidonic acid )*'+,- Based on many comprehensive studies$ the level of one representative isoprostane$ *.'F!t'isoprostane /formerly denoted 0' iso'prostaglandin F!!1$ in blood or urine is widely regarded as the 2gold standard3 biomarker for the assessment of oxidative stress )&'#,- everal methods have been developed for the quanti(cation of *.'F!t'isoprostane$ including GC4MS )0$5,$ LC4MS )*"$**,$ RIA )*!$*+, and ELISA )*&'*%,$ and all of these methods are widely employed for the assessment of systemic oxidant stress- However$ in Figure *- Schematic representation of pathways for the formation$ metabolism and excretion of isoprostanes in vertebrates- Multiple pathways for isoprostane metabolism have been identi(ed$ including "'oxidation$ glucuronidation and the formation of glutathione adducts- Analysis of IsoP'M and4or pretreatment of urine samples with "'glucuronidase can provide insights into variations in metabolism among individuals due to genetics$ diet$ lifestyle$ environment and4or disease-

2 2 contrast to enzymatically'derived eicosanoids$ *.'F!t'isoprostane concentrations determined by GC4 MS or LC4MS do not always correlate well with those obtained using immunoassay methods- Some studies have reported good correlation among these methods )*&$*.,$ whereas others have not )*#,- urther$ there is limited but clear evidence that isoprostanes are rapidly and extensively metabolized in humans )*0'!",- Given the need to rapidly clear these potentially toxic substances from the body$ this is not unexpected- Although one metabolite has been identi(ed and can be independently quanti(ed )!*,$ there appear to be multiple metabolic mechanisms for isoprostane metabolism- Since even relatively small elevations in isoprostane levels have been reported to be a signi(cant risk indicator for cardiovascular disease )!!,$ consideration of the impact of isoprostane metabolism on results obtained by various assay methods is important /Fig- *1- - GC4MS and LC4MS methods typically quantify *.'F!t'isoprostane$ but not isoprostane metabolites- However$ the values obtained can be dependent on the sample preparation protocol )!+,- For example$ four separate isoprostanes contribute to the GC4MS peak if solid phase extraction and TLC are employed for sample preparation- However$ if immunoa6nity chromatography is employed to remove interfering substances$ then only *.'F!t'isoprostane is present in the GC4MS peak- A simple one'step method for immunoa6nity puri(cation of *.'F!t'isoprostane$ which can then be analyzed by GC4MS$ LC4MS or ELISA has recently been published )*",- Convenient and a7ordable columns for this application are now available from Oxford Biomedical Research /see Table on Page.1- - Given the large number of isoprostanes generated from the action of reactive oxygen species on arachidonic acid in vivo$ and the likelihood that multiple *.'F!t'isoprostanes and isoprostane metabolites undoubtedly contribute to 2*.'F!t'isoprostane2 values determined by immuno' assays$ with di7erences among immunoassays expected based on the relative speci(city of the antibodies employed )!",- The antibody employed in our ELISA kit has been extensively characterized so that *.'F!t'isoprostane results obtained by ELISA for serum Figure!- The e7ect of pretreatment with "'glucuronidase on the concentration of F!'isoprostanes in pooled human urine samples- Human urine collected from % normal individuals was separated into two pools- One pool was spiked with *" ng4ml of synthetic *.'F!t'IsoP- Samples were analyzed using standard published protocols with /9G1 or without pretreatment with "'glucuronidase- PBS represents values obtained for phosphate bu7ered saline preincubated with the same quantity of "'glucuronidase- samples following established solid phase extraction protocols 8 or following immunoa6nity isolation of *.'F!t'isoprostane ' correlate very well with those results obtained by GC4MS )*&$*.,- - Indeed$ given the rapid and extensive metabolism of isoprostanes in vivo$ including "'oxidation$ glucuronidation and other pathways )*0$!&,$ and the well documented inter'individual di7erences that have been reported for at least some of these pathways )!#$!0,$ it is actually pretty amazing that isoprostane assays have emerged as a 2gold standard3 for oxidative stress- Further complicating comparison of isoprostane values obtained by di7erent analytical techniques are the signi(cant di7erences among the methods for sample preparation /e-g- multiple Sep'Pak 9 TLC versus immunoa6nity1- - Assays have been developed to quantify one major *.'F!t'isoprostane metabolite$!$+'dinor'0'iso'pgf!! )*.$ *0,$ a7ording the opportunity to evaluate and factor in inter'individual di7erences in metabolism by one pathway of this isoprostane- To facilitiate these e7orts$ immunoa6nity columns speci(c for this metabolite /!$+'dinor'0'iso'pgf!! : IsoP'M1 are now available from Oxford Biomedical Research /see Page.1- - In addition$ the rapid and extensive metabolism of *.'F!t'isoprostane$ suggests that elevated isoprostane levels best serve as biomarkers for acute oxidative

3 Figure +- Immunohistochemical localization of isoprostanes in the brain of Alzheimer<s Disease /AD1 patients /left1- Neurons in the hippocampus of AD patients stain intensely in (xed sections

3 3 Figure +- Immunohistochemical localization of isoprostanes in the brain of Alzheimer<s Disease /AD1 patients /left1- Neurons in the hippocampus of AD patients stain intensely in (xed sections treated with anti'*.'f!t'isoprostane- Results obtained for an age'matched control are shown in the right (gure- Courtesy of S- Basu- See ref-!% for additional data- stress- In animal models$ the pronounced elevation of *.'F!t'isoprostane in response to oxidative stress returns to baseline values within!& hours )!$5,- The strong correlations reported between *.'F!t'isoprostane levels and conditions such as cardiovascular disease in humans is presumably due to chronic oxidative stress to replenish the rapidly metabolized *.'F!t'isoprostane- - Although GC4MS or LC4MS may more reliably quantify levels of a speci(c isoprostane$ e-g- *.'F!t'isoprostane$ the ability of immunoassays to detect isoprostane metabolites )*5,$ and the more robust assessment of isoprostane production that can be obtained by pretreatment of urine with "'glucuronidase may further improve the utility of isoprostanes as a biomarker for oxidative stress by making the measurements more independent of variations in metabolism- - Glucuronidation is a major pathway for isoprostane metabolism: With the exception of!"'hete )!.,$ glucuronidation is not an important pathway for the excretion of enzymatically'derived eicosanoids- Based on the signi(cant di7erences between the stereochemistry of isoprostanes and prostaglandins$ and on the observation that approximately."; of radiolabeled *.'F!t'Isoprostane elutes with the aqueous fraction during solid phase extraction of human urine )*0,$ we investigated the extent to which isoprostanes are excreted as glucuronic acid conju'gates in humans- Whether quanti(ed by GC4MS$ RIA or ELISA )!&$!%,$ pretreatment of human urine samples with "'glucuronidase increased the *.'F!t' Isoprostane levels by an average of =*"";$ indicating that glucuronidation is an important pathway for *.'F!t' isoprostane elimination /Fig-!1- Moreover$ the extent of glucuronidation ranged from!0; to 0"; for the human urine specimens examined- This is not surprising based on well'known inter'individual di7erences in the expression of UDP'glucuronyl transferases and the impact of diet$ lifestyle and other factors on the expression of these enzymes )!#$!0,- Given the extent of and the wide variations observed for *.'F!t'Isoprostane glucuronidation$ it is strongly recommended that urine specimens be pretreated with "'glucuronidase prior to isoprostane analysis to provide more accurate assessment of oxidative stress- Our new immunoa6nity columns speci(c for *.'F!t'Isoprostane /IsoP1 and!$+'dinor'0'iso'pgf!! /IsoP'M1 are recommended for the preparation of serum or urine samples for analysis 8 whether by GC4MS$ LC4MS or ELISA- ur puri(ed antibodies to isoprostanes and IsoP'M can also be used for immunohistochemical localization of these biomartkers for oxidative damage )!%,- Indeed$ *.'F!t'Isoprostane was recently used to visualize the high concentrations of IsoP in the brain of Alzheimer<s patients /Fig- +1- Since$ as detailed above$ the values obtained for the concentration of IsoP in serum or urine samples depend on /a1 the method for sample preparation$ /b1

4 4 the analytic method$ and /c1 the rate of isoprostane metabolism in the subjects- In order to assist investigators who wish to compare results obtained using di7erent sample preparation and analytical methods$ we have prepared a large pool of normal human urine and have obtained IsoP concentrations using SepPak isolation and GC4MS as well as by ELISA using a proprietary extraction' free method- Values were obtained? "'glucuronidase pretreatment- A second pool of urine has been prepared by spiking with authentic *.'F!t'isoprostane to provide an elevated calibrator- Additional e7orts to further standardize and improve isoprostanes as biomarkers for oxidative stress are ongoing- However$ it is critical that investigators in this (eld be cognizant of the impact of experimental methods and IsoP metabolism as they design and execute their studies- *- Morrow J D$ Harris T$ Roberts LJ- /*55"1 Noncyclooxygenase oxidative formation of a series of novel prostaglandins: analytical rami(cations for measurement of eicosanoids- Anal- Biochem-$ *&:*'*"!- Morrow JD$ Hill K$ Burke R F$ Nammour TM$ Badr K F$ Roberts LJ$ /*55"1 A series of prostaglandin F!'like compounds are produced in vivo in humans by a non'cyclooxygenase$ free radical'catalyzed mechanism- Proc Natl Acad Sci USA- 0#:5+0+'5+0#- +- Morrow JD$ Roberts L-J- /*55#1 The isoprostanes: unique bioactive products of lipid peroxidation- Prog Lipid Res-$ +%:*'!*- &- Montuschi P$ Barnes P$ Roberts LJ- /!""#1 Insights into oxidative stress: the isoprostanes- Curr Med Chem-$ *&:#"+'*#-.- Morrow JD- /!""%1 The isoprostanes ' unique products of arachidonate peroxidation: their role as mediators of oxidant stress- Curr Pharm Des-$ *!:05.'5"!- %- Cracowski JL$ Durand T- /!""%1 Cardiovascular pharmacology and physiology of the isoprostanes- Fundam Clin Pharmacol-$!":&*#'!#- #- Morrow JD$ Roberts L-J- /*5551 Mass Spectrometric Quanti(cation of F!'Isoprostanes in Biological Fluids and Tissues as Measure of Oxidant Stress- Meth Enz-$ +"":+'*!- 0- Morrow JD$ Awad JA$ Boss HJ$ Blair IA$ Roberts LJ- /*55!1 Non'cyclooxygenase'derived prostanoids /F!'isoprostanes1 are formed in situ on phospholipids Proc Natl Acad Sci-$ USA 05$*"#!*' *"#!.- 5- Liang Y$ Wei P$ Duke RW$ Reaven PD$ Harman SM$ Cutler RG$ Heward CB- /!""+1 Quanti(cation of 0'iso'prostaglandin'F!! and!$+'dinor'0'iso' prostaglandin'f!! in human urine using liquid chromatography'tandem mass spectrometry- Free Radical Biol- Med-$ +&:&"5'&*0- *"- Sircar D$ Subbaiah PV- /!""#1 Isoprostane Measurement in Plasma and Urine by Liquid Chromatography8Mass Spectrometry with *'Step Sample Preparation Clinical Chemistry.+$.+/!1:!.*' 0 - **- Helmersson J$ Basu S- /*5551 F!'isoprostane excretion rate and diurnal variation in human urine- Prost- Leuk- Essen Fatty Acids %*:!"+'!".- *!- Basu S- /*5501 Radioimmunoassay of 0'iso' prostaglandin F!!: an index for oxidative injury via free radical catalyzed lipid peroxidation- Prostaglandins Leukot Essent Fatty Acids-.0:+*5' +!.- Product Description Price EA 0& Isoprostane ELISA kit for serum$ cell extracts$ etc >!05-"" EA 0. Extraction'free Isoprostane ELISA kit for urine >!05-"" IS +" *.'F!t'Isoprostane /IsoP1 immunoa6nity spin columns >*""-"" IS &"!$+'dinor'0'iso'pgf!! /IsoP'M1 immunoa6nity spin columns >*!.-"" IS!" Goat anti'*.'f!t'isoprostane /anti'isop1 puri(ed IgG >*+*-"" GL 0. "'Glucuronidase pretreatment kit for IsoP analyses >0.-"" IS." Normal 9 elevated human urine IsoP calibrators >..-"" CR "* Urinary Creatinine Assay Kit /to normalize IsoP values1 >05-""

5 5 *+- Sasaki DM$ Yuan Y$ Gikas K$ Taber D$ Morrow JD$ Roberts LJ$ Callewaert DM- /*5551 An immunometric ELISA for *.'F!t'Isoprostane$ an urinary biomarker for oxidant stress- Free Radical Biol- Med--!#:S&+- *&- Proudfoot J$ Barden A$ Mori TA$ Burke V$ Croft KD$ Beilin LJ$ Puddey IB- /*5551 Measurement of urinary F! isoprostanes as markers of in vivo lipid peroxidation 8 A comparison of enzyme immunoassay with gas chromatography4mass spectrometry Anal Biochem-$!#!:!"5'!*.- *.- Roberts LJ$ Moore KP$ Zackert WE$ Oates JA$ Morrow JD- /*55%1 Identi(cation of the major urinary metabolite of the F!'isoprostane 0'iso'prostaglandin F!! in humans J- Biol Chem-$!#*:!"%*#'!"%!"- *%- Basu S- /*5501 Metabolism of 0'iso'prostaglandin F!!- FEBS Lett-$ &!0:+!'+%- *#- Chiabrando C$ Valagussa A$ Rivalta C$ Durand T$ Guy A$ Zuccato E$ Villa P$ Rossi JC$ Fanelli R- /*5551 Identi(cation and measurement of endogenous beta'oxidation metabolites of 0'epi'Prostaglandin F!!- J Biol Chem-$!#&:*+*+'*+*5- *0- Morrow J$ Zackert W$ Yang J$ Kuhrts E$ Callewaert DM$ Taber D$ Oates J$ Roberts LJ- /*5551 Quantitation of the Major Urinary Metabolite of the Isoprostane *.'F!t'Isoprostane /0'iso'PGF!a1 by a Stable Isotope Dilution Mass Spectrometric Assay- Analytical Biochem-$!%5:+!%'++*- *5- Schwedlhelm E$ Bartling A$ Lenzen H$ Tsikas D$ Maas R$ Brummer J$ Gutzki F'M$ Chem I$ Berger J$ Frolioch JC$ Boger RH- /!""&1 Urinary 0'iso' prostaglandin F!! as a risk marker in patients with coronary heart disease: a matched case'control study- Circulation *"5:0&+'0&0-!"- Tsikas D$ Schwedhelm E$ Suchy MT$ Niemann J$ Gutzki FM$ Erpenbeck VJ$ Hohlfeld JM$ Surdacki A$ Frolich JC- /!""+1 Divergence in urinary 0'iso' PGF!!- /ip F!!'III$ *.'F!t'IsoP1 levels from gas chromatography'tandem mass spectrometry quanti(cation after thin'layer chromatography and immunoa6nity column chromatography reveals heterogeneity of 0'iso'PG F! Possible methodological$ mechanistic and clinical implications- J Chromatogr B Analyt Technol Biomed Life Sci-$ #5&:!+#'!..-!*- Callewaert DM$ McGowen R$ Sloan C$ Godschalk K$ Basu S$ Morrow J$ Gupta SV- /!"".1 Isoprostane Glucuronides in Human Urine and the Evaluation of Oxidative Stress- Free Radical Biol- Med-$ +5: S**"-!!- Prakash C$ Zhang JY$ Falck JR$ Chauhan K$ Blair IA- /*55!1!"'Hydroxyeicosatetraenoic acid is excreted as a glucuronide conjugate in human urine- Biochem Biophys Res Commun-$ *0./!1:#!0'++-!+- Callewaert D$ Sloan C- Enzyme Immunoassay of Isoprostanes in Free Radicals and Antioxidant Protocols$! nd Ed- /R- Pryor$ S- Murthy and R- Uppu$ eds1 Methods in Molecular Biology /in press1-!&- Wells PG$ Mackenzie PI$ Chowdhury JR$ Guillemette C$ Gregory PA$ Ishii Y$ Hansen AJ$ Kessler FK$ Kim PM$ Chowdhury NR$ Ritter JK- /!""&1 Glucuronidation and the UDP'glucuronosyltransferases in health and disease Drug Metab Dispos-$ +!/+1:!0*'5"!.- Miners JO$ McKinnon RA$ Mackenzie PI$ /!""!1 Genetic polymorphisms of UDP' glucuronosyltransferases and their functional signi(cance- Toxicology- *0*'*0!:&.+'%-!%- Casadesus G$ Smith MA$ Basu S$ Hua J$ Capobianco DE$ Siedlak SL$ Zhu X$ Perry G- /!""#1 Increased isoprostane and prostaglandin are prominent in neurons in Alzheimer disease- Mol Neurodegener-$!:!!#- Blair IA- /!""%1 Endogenous glutathione adducts- Curr Drug Metab-$ Dec; #/01:0.+'#!-

Isoprostanes: Biomarkers for Oxidative Stress

Isoprostanes: Biomarkers for Oxidative Stress Denis Callewaert, Ph.D. President, Oxford Biomedical Research, Inc. Professor Emeritus, Oakland University 2012 Oxford Biomedical Research, Inc. All rights