Virtual Free Radical School Measurement of F 2

Virtual Free Radical School Measurement of F 2 -Isoprostanes and Isofurans as Indices of Oxidative Stress Status Jack Roberts , Josh Fessel, and Jason Morrow Vanderbilt University L. Jackson Roberts, II, MD, Dept. Pharmacology, Vanderbilt University, Nashville, TN 37232 Tel: 615 -343 -1816, Fax: 615 -343 -9446, EM: jack. roberts@vanderbilt. edu F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 1

Virtual Free Radical School Measurement of F 2 -Isoprostanes and Isofurans as Indices of Oxidative Stress Status Jack Roberts Jason Morrow F 2 -Isoprostanes Josh Fessel Society For Free Radical Biology and Medicine Roberts et al. 2

Free Radicals in Human Disease • • Thought to play a causative role in a wide variety of human diseases ranging from cancer, neurodegenerative disease, atherosclerosis, and even the normal processin translating However, a majoraging impediment these hypotheses to fact has been the lack of a reliable approach to assess oxidative stress status – Thisinis humans because most methods developed a lack in specificity and/or sensitivity or are too invasive for human investigation F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 3

“One of the greatest needs in the field now is the availability of a non-invasive test to probe the oxidative stress status of humans” William A. Pryor, Co-Editor-In Chief Free Radical Biology & Medicine, 1989 F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 4

F 2 -Isoprostanes Discovered One Year Later They are: • • PGF 2 -like compounds produced nonenzymatically by free radical-induced peroxidation of arachidonic acid (AA) Produced in abundance in vivo in quantities far exceeding cyclooxygenase-derived PGs Capable of exerting potent biological actions 4 regioiosmers are formed (5 -, 8 -, 12 -, and 15 series), each of which are comprised of 8 racemic diastereomers F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 5

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 6

Attributes of Measuring F 2 -Iso. Ps as a Biomarker of Lipid Peroxidation • • Specific products of lipid peroxidation Very stable Detectable in all normal biological fluids and tissues – Thus allowing the definition of a normal range NICI GC/MS assay has a lower limit of detection in the low pg range, a precision of 6%, and an accuracy of 96% F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 7

Attributes of Measuring F 2 -Iso. Ps as a Biomarker of Lipid Peroxidation • Their formation increases dramatically in animal models of oxidant injury • Their formation is modulated by antioxidant status – Impairment of endogenous antioxidants enhances their formation – Administration of antioxidant agents suppresses their formation F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 8

An Important Concept Regarding the Formation and Measurement of F 2 Iso. Ps • Initially formed in situ on phospholipids (PL) and then released by phospholipase action • • Levels of free F 2 -Iso. Ps, e. g. in plasma, provides an index of total endogenous production of Iso. Ps Levels of esterified F 2 -Iso. Ps can localize oxidant injury in key tissues/organs of interest F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 9

TBARS Assay for MDA: The Most Widely Used Measure of Lipid Peroxidation • Popular – Because it can even be performed by the intellectually impaired • Problems – The TBARS assay is not specific for MDA – MDA is not a specific product of lipid peroxidation F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 10

Comparison of Measurements of F 2 Iso. Ps and MDA • • In vitro during oxidation of rat liver microsomes In vivo in rat liver following administration of CCl 4 to induce a severe oxidant injury to the liver: F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 11

Comparison of Measurements of F 2 -Iso. Ps and MDA In Vitro In Vivo The formation of both in vitro correlate well - note there is ~25, 000 times more MDA than F 2 -Iso. Ps F 2 -Isoprostanes However, in vivo levels of F 2 -Iso. Ps detected are ~28 -fold higher than MDA Society For Free Radical Biology and Medicine Roberts et al. 12

An Example of the Value of Measuring F 2 -Iso. Ps • • The free radical theory of aging is a very popular theory However, indices of oxidative stress measured in young and old animals, e. g. MDA and protein carbonyls, have in most cases yielded conflicting or unimpressive differences (only 2 - to 3 -fold or less) F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 13

Plasma Concentrations of F 2 -Iso. Ps in Young and Aged Rats Measurement of F 2 Iso. Ps unveils profound oxidant stress in aged rats Roberts LJ, II & Reckelhoff JA BBRC 287: 254, 2001 F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 14

Limitations of Measuring Unmetabolized F 2 -Iso. Ps • • Does not provide a time-integrated index of Iso. P production must be taken to prevent formation of Precautions Iso. Ps ex vivo by autoxidation in some biological fluids, e. g. plasma, during storage • Unmetabolized Iso. Ps in urine can, in part, reflect renal rather than systemic production of Iso. Ps F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 15

Advantages of Measuring a Urinary F 2 Iso. P Metabolite • • Can provide a time-integrated index of total endogenous Iso. P production by collecting urine over several in hours – Important situations where there are large fluctuations in the extent of oxidant injury over time Measured in urine – Eliminates need for blood collection • Metabolites cannot be formed ex vivo by autoxidation – No special storage conditions are required F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 16

Metabolic Fate of 15 -F 2 t-Iso. P (8 -Iso-PGF 2 ) in Humans -oxidation 5 -reduction Major urinary metabolite A GC/MS assay for F 2 -Iso. PM has been developed (Roberts LJ, II, et. al. J. Biol. Chem : 271: 20617, 1996) F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 17

Assessments of F 2 -Iso. P Production in Patients With Polygenic Hypercholesterolemia Plasma Esterified Plasma Free Urinary F 2 -Iso. PM The relative increases in F 2 -Iso. Ps assessed by all 3 approaches are in close agreement F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 18

Partial List of Disorders in Which Measurements Of F 2 -Iso. Ps Has Implicated a Pathogenic Role for Oxidative Stress • • • Smoking Alzheimer’s disease Hunington’s disease Atherosclerosis Hepatorenal syndrome Scleroderma Cardiac/renal ischemia/reperfusion injury Coronary angioplasty Se and vitamin E deficiency Organophosphate poisoning Hyperhomocysteinemia Renal transplant dysfunction F 2 -Isoprostanes • • Diabetes • • Halothane hepatotoxicity Rhabdomyolysis Bile duct obstruction O 2 pulmonary toxicity in premature infants Acetaminophen poisoning Age-related decline in renal function Cr (VI) poisoning Retinopathy of the newborn Alcohol ingestion Paraquat poisoning Cisplatin-induced renal dysfunction Society For Free Radical Biology and Medicine Roberts et al. 19

Potential Limitation of Measuring F 2 -Iso. Ps As An Index of Oxidative Stress • An intermediate in the formation of Iso. Ps is a carbon-centered radical [C ] • Intramolecular attack by this radical is required to form the cyclopentane ring in Iso. Ps • Competing with this is attack of the [C ] by molecular O 2 + O ] to form other products [C COO 2 Accordingly, measurement of F -Iso. Ps is an • 2 insensitive marker of lipid peroxidation in setting of high O 2 tension, e. g. hyperoxic lung injury F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 20

Attack of the C • in the Iso. P Pathway by O 2 Leads to the Formation of “Other Products” • • • F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 21

Discovery of New Products of Lipid Peroxidation • We recently discovered the formation of a series of compounds with a substituted tetrahydrofuran ring (Isofurans) (Fessel JP & Roberts LJ, II, et. al. submitted) which are products of oxygen attack on the carbon centered radical Iso. Pbecomes pathway. highly – Whereas the formation of in F the -Iso. Ps 2 disfavored at elevated oxygen tensions, the formation of Iso. Fs becomes highly favored F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 22

Formation of Isofuran Regioisomers • • F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 23

Effect of O 2 Tension on the Formation of F 2 -Iso. Ps and Iso. Fs During Oxidation of Arachidonic Acid In Vitro The formation of Iso. Fs becomes highly favored at high concentrations of O 2 F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 24

The Ratio of Iso. Fs to F 2 -Iso. Ps Correlates with Tissue Oxygenation In Vivo Levels of Iso. Fs are higher than Iso. Ps in highly oxygenated organs (brain and kidney) but lower in poorly oxygenated organs (liver) F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 25

Summary • • The formation of F 2 -Iso. Ps and Iso. Fs are differentially modulated by oxygen tension Accordingly, combined measurement of both F 2 Iso. Ps and Iso. Fs may provide the best assessment of lipid peroxidation and oxidative stress status F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 26