Lipid Peroxidation (F2-Isoprostanes)

Description

Lipid peroxidation is one type of oxidative damage that has been extensively studied with respect to age. The allylic hydrogens in the polyunsaturated fatty acid components of phospholipids in cellular membranes make membranes extremely sensitive to free radical oxidation. One of the major problems confronting investigators measuring lipid peroxidation is the accuracy of the assays measuring lipid peroxidation by malondialdehyde (MDA) and 4-hydroxy-2-nonenol (HNE) levels.  The Roberts laboratory was the first to describe the non-enzymatic production of a group of prostaglandin-like compounds that arise from free radical attack on membrane phospholipids (Roberts and Morrow, 2000).  As shown in Figure 1, arachadonic acid moieties, esterified to membrane phospholipids, are converted to compounds isomeric to prostaglandin F and are referred to as F2-isoprostanes (F2-isoPs).  Esterified F2-isoPs are released from the membrane, by the action of phospholipase, into the blood stream and are ultimately excreted in the urine (Morrow et al., 1999).  Thus, plasma levels of free F2-isoPs provide a measure of the total endogenous production of F2-isoPs from all sites in the body (11).  The formation of F2-isoPs provides an excellent marker of oxidative stress because they can be measured with precision down to the picomolar level; they are relatively stable compounds; they do not exhibit diurnal variations; they are present in detectable quantities in all normal biological tissues; and lastly, F2-isoPs levels are modulated by the antioxidant status of the organism but are not affected by the lipid composition of the diet (Roberts and Morrow, 2000; Janssen, 2001).  

Measurement of plasma free F2-isoPs levels provides one with an assay to assess total endogenous F2-isoPs production, i.e., an excellent measure of the level of oxidative stress of the organism. Measurement of tissue levels of esterified F2-isoPs provides additional insight into the total oxidative burden of the organism and, in addition, enables one to determine whether one tissue is more susceptible to oxidative damage than another tissue. Roberts and Reckelhoff (2001) were the first to measure the effect of age on F2-isoP levels.  They showed that plasma levels of free and esterfied F2-isoPs were dramatically increased with age in male Sprague Dawley rats.  As shown in Figure 2, our group subsequently showed that F2-isoPs levels increase with age in the plasma, and also in liver and kidney tissue of male F344 rats and that these increases are modulated by dietary restriction (Ward et al., 2005). Furthermore, the changes in F2-isoPs levels in liver and kidney tissue are directly correlated with oxidative damage to DNA.  

References:

Janssen, L.J. (2001). Isoprostanes: an overview and putative roles in pulmonary pathophysiology. Am. J. Physiol. Lung Cell Mol. Physiol. 280, L1067-L1082.

Morrow, J.D., Chen, Y., Brame, C.J., Yang, J., Sanchez, S.C., Xu, J., Zackert, W.E., Awad, J.A. and Roberts II, L.J. (1999). The isoprostanes: unique prostaglandin-like products of free-radical-initiated lipid peroxidation. Drug Metab. Rev. 31, 117-139.

Morrow, J.D. and Roberts II, L.J. (1997). The isoprostanes: unique bioactive products of lipid peroxidation. Prog. Lipid Res. 36, 1-21.

Roberts II, L.J., Morrow, J.D. (2000). Measurement of F2-isoprostanes as an index of oxidative stress in vivo. Free Rad. Biol. Med. 28, 505-513.

Roberts II, L.J. and Reckelhoff, J.F. (2001). Measurement of F2-isoprostanes unveils profound oxidative stress in aged rats. Biochem. Biophys. Res. Comm. 287, 254-256.

Ward, W.F., Qi, W., Van Remmen, H., Zackett, W.E., Roberts II, L.J., and Richardson, A. (2005). Effects of age and caloric restriction on lipid peroxidation: measurement of oxidative stress by F2-isoprostane levels. J. Gerontol. A Biol. Sci. Med. Sci.  60, 847-851.

Experimental Methods

The levels of F2-isoprostanes (8-iso-PGF2) are measured using the GC/MS procedure developed by Roberts and Morrow (2000) and described by our group (Ward et al., 2005). 

F2-isoprostanes and isofurans will be measured in tissues using gas chromatography-mass spectrometry methods as previously described (Morrow and Roberts, 1994). The isoprostane/isofuran level can be measured in samples of 100 mg of tissue, 1 ml of plasma (requires sample collection from 3-4 mice or 1 rat) or 300 ul of urine.  Tissue is homogenized in chloroform:methanol containing BHT (0.005%) to prevent auto-oxidation, dried under a stream of nitrogen, and re-suspended in methanol containing BHT.  Esterified F2-isoprostanes (and F2-isofuranes) in phospholipids are saponified by adding aqueous potassium hydroxide (this frees fatty acids from lipids). The sample is acidified and diluted with water.  Deuterated-F2-isoprostane internal standard is then added to the mixture. For the measurement of free F2-isoprotanes/F2-isofuranes in plasma, the extraction and hydrolysis steps are omitted, and the sample is simply acidified, diluted, and the internal standard added.  The mixture is subsequently run on a silica column to separate isoprostanes/isofuranes from bulk fatty acids.  The eluate is converted to pentafluorobenzyl esters, by treatment with pentafluorobenzyl bromide (this step is necessary because free fatty acids are difficult to separate by gas chromatography).  The mixture is subjected to thin layer chromatography to remove the excess pentafluorobenzyl bromide and unreacted fatty acids.  The F2-isoprotane/isofurane fraction is extracted using ethyl acetate, and will be analyzed by injection into a Thermo Finnigan TRACE DSQ single quadrupole mass spectrometer.  This instrument is capable of electron impact and chemical ionization, with positive and negative ion detection.  The F2-isoprostanes and isofurans are quantified by peak height, and the data is corrected with the internal standard and expressed as nanogram of F2-isoprostanes and F2-isofuranes per mL of plasma or per gram tissue. 

References:

Roberts II, L.J., Morrow, J.D. (2000). Measurement of F2-isoprostanes as an index of oxidative stress in vivo. Free Rad. Biol. Med. 28, 505-513.

Ward, W.F., Qi, W., Van Remmen, H., Zackett, W.E., Roberts II, L.J., and Richardson, A. (2005). Effects of age and caloric restriction on lipid peroxidation: measurement of oxidative stress by F2-isoprostane levels. J. Gerontol. A Biol. Sci. Med. Sci.  60, 847-851.

Sample Preparation Guidelines

  • Tissue Samples:  We require snap frozen tissue stored at -80oC for the F2-isoprostane assay.  For most tissues, 100-150 mg of tissue is optimal for analysis.  
  • Cells:  We a need a cell pellet harvested from 2-4 million washed in PBS, frozen in liquid nitrogen and stored at -80oC. 
  • Blood:  We require ~1 ml of blood/plasma for the F2-isoprostane assay.  We routinely collect blood from the inferior vena cava of anesthetized animals into pre-chilled heparin-coated tubes.  The tubes are centrifuged at 1,500 x g for 10 min at 4o C to give plasma, which is flash-frozen in liquid nitrogen and stored at -80oC.  Normally, enough blood can be collected from one rat for the F2-isoprostane assay; however, for mice blood needs to be pooled from 2 to 4 mice to have enough sample for optimal analysis.