f 2 -isoprostanes society for free radical biology and medicine roberts et al. 1 measurement of f 2...

F2-Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 1

Measurement of F2-Isoprostanes and Isofurans as Indices of Oxidative Stress Status

Jack Roberts , Josh Fessel, and Jason Morrow

Vanderbilt University

Virtual Free Radical School

L. Jackson Roberts, II, MD, Dept. Pharmacology, Vanderbilt University, Nashville, TN 37232 Tel: 615-343-1816, Fax: 615-343-9446, EM: [email protected]


Measurement of F2-Isoprostanes and Isofurans as Indices of Oxidative Stress Status

Jack Roberts Josh Fessel Jason Morrow

Virtual Free Radical School


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 aging process

• However, a major impediment in translating these hypotheses to fact has been the lack of a reliable approach to assess oxidative stress status in humans

– This is because most methods developed a lack in specificity and/or sensitivity or are too invasive for human investigation


“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


F2-Isoprostanes Discovered One Year Later

• PGF2-like compounds produced non-enzymatically 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

They are:


AACOOH

COOH

.

-H

.

O2

O

COOH

O

O

O COOH

.O2

O

O COOH

OOH

15

[H]

COOH

OHOH

OH

..COOHCOOH

.

.

.

8125

5-Series 12-Series 8-Series

[H] [H] [H]

COOH

OHOH

OH OH

OH

OHCOOH

OHCOOH

OH

OH

O2O2O2

O

O

O

O

O

O

OOHCOOHCOOH

OOH

OOHCOOH

O

O COOHO

O

COOH

O

O COOH

O2O2O2

O

COOH

O

OOO

COOHO

COOH

..

.

15-Series


Attributes of Measuring F2-IsoPs 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%


Attributes of Measuring F2-IsoPs 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


An Important Concept Regarding the Formation and Measurement of F2-IsoPs

• Initially formed in situ on phospholipids (PL) and then released by phospholipase action

PL-AA PL-IsoP Free IsoP Oxidation Lipase

• Levels of free F2-IsoPs, e.g. in plasma, provides an index of total endogenous production of IsoPs

• Levels of esterified F2-IsoPs can localize oxidant injury in key tissues/organs of interest


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


Comparison of Measurements of F2-IsoPs and MDA

• In vitro during oxidation of rat liver microsomes• In vivo in rat liver following administration of CCl4 to induce a severe oxidant injury to the liver:

CCL4

P-450

CCL3 .LH

L . O2 LOO.


Comparison of Measurements of F2-IsoPs and MDA

Time (min)

MD

A (

nmol

/mg

prot

ein)

F2 -IsoP

s (pmol/m

g protein)0 10 20 30 40 50

0

20

40

60

80

100MDA F2-IsoPs

0

1

2

3

4

5

In Vitro

The formation of both in vitro correlate well - note there is ~25,000 times more MDA than F2-IsoPs

Fol

d-In

crea

se A

bove

Bas

elin

e

0

2

450

60

70

80

90

100

F2-IsoPs SGPT MDA

In Vivo

However, in vivo levels of F2-IsoPs detected are ~28-fold higher than MDA


An Example of the Value of Measuring F2-IsoPs

• 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)


Fre

e F 2

-Iso

Ps

(pg/

ml)

0

300

600

900

1200

1500

1800

Young Aged

(3-4 mos) (22-24 mos)

4.3-fold

42.9-fold

20.3-fold

Free F2-IsoPs

Roberts LJ, II & Reckelhoff JA BBRC 287:254, 2001

Plasma Concentrations of F2-IsoPs in Young and Aged Rats

Measurement of F2-IsoPs unveils profound oxidant

stress in aged rats


Limitations of Measuring Unmetabolized F2-IsoPs

• Does not provide a time-integrated index of IsoP production• Precautions must be taken to prevent formation of IsoPs ex vivo by autoxidation in some biological

fluids, e.g. plasma, during storage

• Unmetabolized IsoPs in urine can, in part, reflect renal rather than systemic production of IsoPs


Advantages of Measuring a Urinary F2-IsoP Metabolite

• Can provide a time-integrated index of total endogenous IsoP production by collecting urine over several hours

• Metabolites cannot be formed ex vivo by autoxidation

– No special storage conditions are required

• Measured in urine

– Eliminates need for blood collection

– Important in situations where there are large fluctuations in the extent of oxidant injury over time


Metabolic Fate of 15-F2t-IsoP (8-Iso-PGF2) in Humans

A GC/MS assay for F2-IsoP-M has been developed

(Roberts LJ, II, et. al. J. Biol. Chem :271:20617, 1996)

COOH

OHOH

OH

OHOH

OHCOOH

-Oxidation 5

-Reduction

15-F2t-IsoP

2,3-Dinor-5,6-Dihydro-15-F2t-IsoP (F2-IsoP-M)

-oxidation 5-reduction

Major urinary metabolite


Este

rifie

d F 2-Is

oPs

(pg/

ml)

0

55

110

165

220

275

Normals Patients

*

* p<0.001

Free

F2-Is

oPs

(pg/

ml)

0

30

60

90

120

Normals Patients

*

* p<0.0001

Plasma Esterified Plasma Free

Assessments of F2-IsoP Production in Patients With Polygenic Hypercholesterolemia

F 2-IsoP

-M (

ng/m

g C

r)

0.0

0.2

0.4

0.6

0.8

1.0

Normals Hi-Chol

*

*p<0.0008

Urinary F2-IsoP-M

The relative increases in F2-IsoPs assessed by all 3 approaches are in close agreement


Partial List of Disorders in Which Measurements Of F2-IsoPs 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

• Diabetes

• Rhabdomyolysis

• Bile duct obstruction

• O2 pulmonary toxicity in premature infants

• Halothane hepatotoxicity

• Acetaminophen poisoning

• Age-related decline in renal function

• Cr (VI) poisoning

• Retinopathy of the newborn

• Alcohol ingestion

• Paraquat poisoning

• Cisplatin-induced renal dysfunction


Potential Limitation of Measuring F2-IsoPs As An Index of Oxidative Stress • An intermediate in the formation of IsoPs is a carbon-centered

radical [C]• Intramolecular attack by this radical is required to form the

cyclopentane ring in IsoPs

• Competing with this is attack of the [C] by molecular O2

[C + O2 COO] to form other products• Accordingly, measurement of F2-IsoPs is an insensitive marker of lipid

peroxidation in setting of high O2 tension, e.g. hyperoxic lung injury


O2

R

RH

AACOOH

COOH

O

COOHO

COOH

O

O

•

•

•

O2

COOH

OO

O

O

Other Products

•

COOH

O

O

COOHO

O

Isoprostanes

•

•

Attack of the C• in the IsoP Pathway by O2 Leads to the Formation of “Other Products”


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 in the IsoP pathway.

– Whereas the formation of F2-IsoPs becomes highly disfavored at elevated oxygen tensions, the formation of IsoFs becomes highly favored


Formation of Isofuran Regioisomers

O COOH

HO

OHOH

O

OH

COOH

OHHO

O

HO

OH

OH

COOHO

OH

COOH

OH

HO

O

HO

OH

COOH

OH

O2

R

RH

AACOOH

COOH

O

COOHO

COOH

O

O

O2

O COOH

HO

OH OH

OCOOH

OH

OHHO

OCOOH

HO

OH

•

•


Effect of O2 Tension on the Formation of F2-IsoPs and IsoFs During Oxidation of Arachidonic Acid In Vitro

0.0

0.3

0.6

0.9

1.2

1.5

g I

soP

s &

Iso

Fs/

mg

AA

1% 21% 100% 1% 21% 100%

Oxygen Tension

IsoPs

IsoFs

The formation of IsoFs becomes highly favored at high concentrations of O2


The Ratio of IsoFs to F2-IsoPs Correlates with Tissue Oxygenation In Vivo

Rat

io I

soF

s/Is

oPs

Brain Kidney Liver0.0

0.8

1.6

2.4

3.2

1.0

Levels of IsoFs are higher than IsoPs in highly oxygenated organs (brain and kidney) but lower in poorly oxygenated organs (liver)


Summary

• Accordingly, combined measurement of both F2-IsoPs and IsoFs may provide the best assessment of lipid peroxidation and oxidative stress status

• The formation of F2-IsoPs and IsoFs are differentially modulated by oxygen tension

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