Isoprostanes: The Rest of the Iceberg

Kirk M. Maxey, Joshua Rokach, Jeff Johnson, and John Lawson

"How could the amount of 11β-PGF_2α in a sample increase by hundreds of pg/ml just by sitting in the freezer for 6 months?" This must have been one of several questions running through the mind of a young student at Vanderbilt University in the late 1980's. His group in the laboratory of Jack Roberts had been using GC/MS to study PGD₂ metabolism, analyzing the m/z 569 ion characteristic of PGF_2α compounds, including the primary plasma PGD₂ metabolite, 11β-PGF_2α. Incredibly, what he found on routine re-analysis of old samples was a marked increase in the 569 ion. Was someone secretly spiking the samples? Was the instrument working properly now? Had it been working before? As the Vanderbilt lab grappled with these questions, the very tip of the isoprostane iceberg began to emerge. The stored samples contained numerous PGF_2α-like compounds, but none of them were 11β-PGF_2α, as seen in Figure 1. None of the known isomers of PGF_2α seemed to match any of these novel compounds—until they tested an obscure isomer, 8-iso PGF_2α, which had been synthesized and sold as an analytical standard by Cayman for many years. 8-iso PGF_2α was definitely present in the samples, but no known mechanism could account for its production as a metabolite of PGD₂. Isoprostanes would soon be regarded as an important marker of in vivo oxidative stress and free radical injury. Ironically, in their biochemical debut they turned up as an artifact induced by the oxidative decomposition of lipids in poorly stored prostaglandin samples. The Vanderbilt group rapidly established several key findings related to their discovery, which still form the basis of isoprostane biochemistry.^1,2

1. There are many, many isoprostanes – clearly more than one hundreddifferent compounds, at least a dozen of which were clearly seen onthe first GC chromatograms.
2. What all isoprostanes have in common, and what distinguishesthem from the better-known prostaglandins, is the fact that thetop (α) and bottom (ω) side-chains are always syn – that is, crowdedtogether next to each other, on the same face of the cyclopentanering. (For a mechanistic explanation of this, see below)
3. Although present, 8-iso PGF_2α is clearly a minor player, representingno more than a few percent of the isoprostane total. (See Figure 2.)
4. All isoprostanes share a non-enzymatic origin via the free radicalinduced oxidation of unsaturated fatty acids.*

* Exceptions include the formation of minor amounts of 8-iso PGF_2α by COX-1 in platelets and by COX-2 in monocytes.^17,47

The minimum requirement for the generation of an isoprostane is a polyunsaturated fatty acid with 3 contiguous, methylene-interrupted double bonds. There are dozens of naturally occurring polyunsaturated fatty acids that meet this requirement, leading to a bewildering array of isoprostane families and regioisomers (see figures on page 3). In spite of this, the isoprostane field has been narrowly focused for nearly a decade on 8-iso PGF_2α, which seems counter-intuitive given its modest contribution to the whole isoprostane family. The reasons for this emphasis were entirely pragmatic: 8-iso PGF_2α was the only isoprostane commercially available in large quantities. 8-Isoprostane EIA kits and deuterated internal standards for 8-iso PGF_2α were introduced by Cayman in 1992, leading to a proliferation of articles about its biology and analysis. A literature search including the period from 1992 to 2000 reveals that fully 90% of isoprostane publications related specifically to 8-iso PGF_2α, and virtually all of these referenced Cayman as the source of either the compound itself, the deuterated internal standard, or the 8-iso PGF_2α EIA kit. Various authors explored the relationship of 8-iso PGF_2α to platelet TP receptors,^4-6 vascular smooth muscle cells,^7-9 coronary arteries,⁸¹ pulmonary artery,¹⁰ cerebral arterioles,¹¹ atherosclerotic placque,¹² intact perfused heart,¹³ rabbit lung,¹⁴ glomerulus,¹⁵ hypoxic alveolar macrophages,¹⁶ monocytes,¹⁷ platelets,^18,19 human semen,^20,21 and myometrium.²² Analytical papers compared GC/MS of 8-iso PGF_2α with EIA,²³ documented increases in 8-iso PGF_2α in smokers,^24-26 experimental brain injury,²⁷ neurodegenerative disease,²⁸ coronary reperfusion,²⁹ and as a general index of oxidative stress.^30,31

However, a handful of the isoprostane publications during this time were pioneering attempts to demonstrate that 8-iso PGF_2α was far from the ideal index marker of the isoprostane family. The abundance of a different regioisomer, iPF_2α-VI, and its facile extraction from lipid samples was demonstrated by Rokach and Fitzgerald.^32-35 Morrow and Roberts showed that A, D, E, and even thromboxane-ring isoprostanes were formed,³⁶ and that isoprostanes from fatty acids other than arachidonate, in particular DHA,³⁷ might be more important markers in the brain.

With the publication of this newsletter, Cayman makes an important step forward in the expansion of the isoprostane field. For the first time, unlabeled and deuterated standards for the isoprostanes in the very important Type IV, V, and VI families will now be commercially available. Isoprostanes from C-18 and C-22 fatty acids will also be introduced, as well as the iPF₃ and iPF₄ isoprostanes derived from the important ω-3 fatty acids EPA and DHA. In short, a new chapter is opening in the field of free radical peroxidation markers. Enzyme immunoassays based on the new molecules are under development and will be introduced in the following months.

Smoking

Ingestion of carbon tetrachloride results in hepatic synthesis of the trichloromethyl radical and massive systemic peroxidative injury followed by death in intact animals. Isoprostane levels in this model increase by over 80-fold.^2,38 The closest voluntary human parallel to this model is the intimate personal fumigation of the human lung with tobacco smoke. While less flagrant than drinking chlorinated hydrocarbons, smoking exposes the lung to a variety of potent oxidants, most notably polyaromatic hydrocarbons (PAH). Isoprostane production (8-iso PGF_2α) as measured by daily urinary excretion is about 2.2 times higher in smokers than in non-smokers.²⁵ Similarly, the urinary concentration of 8-iso PGF_2α is roughly doubled (62 vs. 120 pmol/mmol creatinine) in heavy smokers compared with non-smokers.²⁶ Finally, the same trend is seen in the plasma concentration of 8-iso PGF_2α, with smokers averaging around 570 pmol/l compared to 240 pmol/l for controls.²⁴ Elevations of 8-iso PGF_2α are of particular concern in smokers because of this particular isoprostane's documented vasoconstrictive effects in coronary,³⁹ renal,¹⁵ and pulmonary arteries.¹⁰ Other members of the isoprostane family may be better indicators of oxidative damage in smokers, but few have been evaluated. It should be pointed out that lung surfactant is comprised of a carefully modulated lipid pool selected for their surface-elastic properties, and arachidonate is not prominent among the polyunsaturated fraction of these lipids. Likewise, it is unknown whether any of the other isoprostanes are biologically active, as virtually none have been systematically tested in physiologic models.

Antioxidant Deficiency

Smokers are known to have depressed levels of the important aqueous antioxidant ascorbic acid (vitamin C), so it is natural to suspect that isoprostanes might be a general indicator of antioxidant deficiency. Studies attempting to correlate antioxidant levels (ascorbate, tocopherols, α-carotene, and others) with plasma or urinary 8-iso PGF_2α have generally failed. This may indicate that none of the isoprostanes correlate with antioxidant insufficiency, or simply that the isoprostane responsible is another, untested isomer. One exception is a study showing that vitamin C supplements decrease F₂-isoprostanes in overweight smokers, but not in those with a normal body mass index (BMI).82 Vitamin E has been shown to reduce 8-iso PGF_2α in Apo-E^-/- mice40 and iPF_2α-VI in LDLR^-/- mice,⁴¹ two animal models of atherosclerosis. It is not clear whether a local excess of free radicals in the atherosclerotic placque, or a generalized deficiency of vitamin E accounts for these findings. Isoprostanes form in the highly structured lipid environment of the membrane, so intuition would lead one to suspect that the ratios between isoprostane family members might be more sensitive indicators than absolute isoprostane quantities. The influence of different lipid environments on the distribution of isoprostane regioisomers has not been studied to date. Finally, all efforts to use isoprostane concentrations to document a benefit from various herbs, vitamin supplements, or other nutritional remedies have thus far failed. This is more likely an indictment of the medicinal value and relative metabolic importance of said remedies than it is of the isoprostanes as a marker of oxidant injury.

Neuronal Oxidative Damage

The brain and retina cannot develop properly without a source of α-3 fatty acids to use for the synthesis of DHA. It is not clear precisely what role peculiar to neuronal function DHA is supporting, but it seems to be a physiochemical one, in that DHA accounts for up to 60% of brain and retinal fatty acid content.⁴² It would be logical, then, to look particularly at the 22-carbon isoprostanes derived from DHA in cerebrovascular, neurodegenerative, and inflammatory central nervous system disorders. Until now, the lack of authentic reference standards for these iPF₄ isoprostanes rendered this impossible. Still, groups studying arachidonate-derived isoprostanes have documented increases in 8-iso PGF_2α and iPGF_2α-VI in Alzheimer's disease (AD), where both isoprostane isomers were elevated about 2-fold in the brain tissue of confirmed AD patients at autopsy.⁴³ 8-iso PGF_2α is also elevated in the CSF of patients with multiple sclerosis.⁴⁴ In traumatic brain injury, a 5-fold increase in 8-iso PGF_2α was demonstrated by EIA using a brain contusion model in rats.²⁷ It would be worthwhile to repeat nearly all of these studies, this time with a focus on the 22-carbon iPF₄ isoprostanes derived from DHA. In addition, a prevalent cause of blindness in the aging western population is the retinal neurodegenerative disease glaucoma. There are at present no good diagnostic biomarkers for the early diagnosis of this disease, and the proximate cause of the frequently elevated intraocular pressure in glaucoma is poorly understood. Isoprostanes, particularly the iPF₄ isoprostanes that are released by degenerating retinal neurons, warrant further examination in both these areas.

Urinary F₂ Isoprostanes

Normal human urine contains 400-500 pg/mg creatinine of 11-dehydro TXB2, which is a good representative marker of a cyclooxygenase-derived eicosanoid. The graph below compares this marker with 4 selected isoprostanes; 8-iso PGF_2α, iPF_2α-VI, 8,12-iso iPF_2α-VI, and 5-epi-8,12-iso iPF_2α-VI. It is immediately clear that the 8,12-iso- iPF_2α-VI family dominates the spectrum of urinary isoprostanes. Little is currently known about isoprostane metabolism following release into the bloodstream as free acid compounds. In the case of the best-studied 8-iso PGF_2α, metabolism via b-oxidation of the a-chain has been reported by two groups.45,46 It is possible that the 5-hydroxyl group in the iPF_2α-VI family inhibits this route of metabolism, leading to increased clearance of the parent isoprostane in the urine.34 Whatever the reasons for this predominance of the iPF_2α-VI family, there is an additional reason for making this isoprostane the marker of choice. Lawson, et al. have devised a much simplified purification procedure that is selective for the iPF_2α-VI family and relies on the easy lactonization-rehydrolysis of the C-1 carboxyl with the adjacent C-5 hydroxyl.³⁵F As sample preparation and purification are the most labor-intensive and technically difficult procedures in most analytical methods for isoprostane analysis, this makes the iPF_2α-VI class especially attractive.

Finally, the issue of 8-iso PGF_2α synthesis by cyclooxygenase (COX) has been raised by at least two groups as a confounding variable when using this isoprostane as a marker of lipid peroxidation.17,47,48 Whether this issue is relevant depends entirely on the intended sample. In urine, the contribution of COX-derived 8-iso PGF_2α is so small as to be inconsequential.26,35 In serum, there is an exhaustive burst of COX activity in platelets as well as, to a lesser extent, the nucleated leukocytes. This metabolism occurs during the normal clotting process, and is greatly accelerated by addition of an ionophore. The amount of 8-iso PGF_2α produced during the normal formation of serum is significant,³⁵ and should be carefully considered when testing supernatants from serum-treated cells, or when serum is the actual sample itself. Care must also be taken when using LPS-stimulated monocytes, as COX-2 in these cells can produce significant amounts of 8-iso PGF_2α.¹⁷

Isoprostanes in Plants

Although almost completely unexplored, isoprostanes can clearly form in plants.49 No other tissue is so intimately exposed to oxidative stress as the plant chloroplast, which manufactures and excretes oxygen while exposed to a dense flux of solar radiation. Plants present a somewhat simplified picture, in that 18-carbon fatty acids dominate plant lipids. The 16 isoprostane isomers resulting from linolenate are illustrated below, and are likely to be present along with other C-18 and C-20 isoprostanes. One of the plant isoprostanes ( 2,3-dinor iPF_2α-III, formed from γ-linolenic acid) is identical to one of the main urinary metabolites of arachidonate-derived 8-iso PGF_2α. The significance of this is unknown, but it shows how complex the isoprostane puzzle can be. It is certainly unclear at this time whether the 2,3-dinor iPF_2α-III present in human urine originates from arachidonate, γ-linolenic acid, or both.

Isoprostanes in Fatty Acid Deficiency

Mead acid is a C-20, trienoic fatty acid which accumulates in essential fatty acid deficiency. The peculiar symptoms of essential fatty acid deficiency include dry, scaly skin, growth retardation, immunocompromise, among others, and are not adequately explained by a lack of eicosanoid signaling. When exposed to oxidants, Mead Acid produces a family of 16 isoprostanes in 2 families, as illustrated below. None have ever been tested for biological activity.

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