A class of natural prostaglandin-like products. The isoprostanes (iPs) are isomeric with another class of natural products, the prostaglandins. The prostaglandins are the result of enzymatic oxygenation of polyunsaturated fatty acids (PUFAs), in particular arachidonic acid (AA). In contrast, the iPs are formed in vivo by a nonenzymatic, free-radical oxygenation of arachidonic acid. This important distinction in the mode of formation of the iPs is responsible for a more complex mixture of iPs being generated in vivo. For example, whereas the endoperoxide prostaglandin G2 (PGG2) is formed by the cyclooxygenase enzymes (COX1 and COX2), four classes of iPs are formed as a result of the free-radical oxygenation of arachidonic acid (Fig. 1), with each class containing 16 iPs for a total of 64 individual iP molecules. The discovery of iPs is important for two reasons: (1) group III iPs are incidental ligands for the prostaglandin receptors; hence they possess biological activity; (2) iPs are the product of oxidative stress. Their measurements have been shown to be a predictor of the onset and severity of inflammatory diseases such as Alzheimer's disease and atherosclerosis. See also: Alzheimer's disease; Circulation disorders; Eicosanoids; Free radical
Mechanism for formation
Two mechanisms for the formation of iPs have been proposed based on in-depth regiochemical and stereochemical analysis of the peroxidation process. These are the endoperoxide mechanism (Fig. 1) and the dioxetane mechanism. Recently, evidence has been reported showing that the dioxetane mechanism may not be operative.
A more detailed mechanistic view for the formation of group VI iPs by the endoperoxide mechanism is presented in Fig. 2. The four iPs shown in the box have been prepared by total synthesis and used in discovery and identification of the same products in human urine (Fig. 4).
A comprehensive nomenclature for iPs has been introduced utilizing the symbols in the prostaglandin nomenclature. The new classification (roman numerals) reflects the use of the ω end of the molecule instead of the COOH in the double-bond counting system—the same way that double bonds in polyunsaturated fatty acids are referred to as ω-3, ω-6, etc. Another nomenclature is also being used for iPs based on the location of the hydroxyl group in the side chain, the carboxyl end being C-1.
Two techniques for measuring iPs are discussed below.
Gas chromatography/mass spectrometry (GC/MS) methods have been developed to quantitate individual iPs, particularly group VI iPs, which appear to be most abundant in urine. Group VI was chosen as the main initial focus because the position of the OH on carbon-5 of the upper side chain allows a lactone to form, which can easily be separated from the rest of the iPs, allowing for a more accurate and reliable determination in biological fluids (Fig. 3).
The GC/MS method outlined in Fig. 3 has enabled measurement of in vitro and in vivo elevated levels of iPF2α-VI in a number of clinical settings, such as ischemia-reperfusion syndromes, hyper-cholesterolemia, Alzheimer's disease, and alcohol-induced liver disease. Elevated levels of iPF2α-VI have been measured in oxidized low-density lipoprotein (LDL) and in aortic atherosclerotic lesions. In all cases, the discovery and identification of iPs of different groups, including those shown in Figs. 2 and 4, have been made possible by the initial availability of authentic material prepared by total synthesis. See also: Gas chromatography; Mass spectrometry
Liquid chromatography tandem mass spectrometry (LC/MS/MS) is another promising method for the identification, separation, and quantitation of iPs. Neither derivatization nor prior purification is required. Arachidonic acid–derived iPs readily generate abundant molecular ions (m/z 353) under ESI (electrospray ionization) conditions in the negative-ion mode, which fragments further to ions, some of which are group-specific (m/z 115, 193, 127, 151). Figure 4 shows the chromatogram of the four groups of arachidonic acid–derived iPs from human urine. The chemical structure in the chromatogram identifies the individual iPs. The four most abundant iPs of urinary group VI have the cis configuration of the side chain. See also: Liquid chromatography; Mass spectrometry
Other novel iPs and isofurans
Reports have shown that oxidation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) result in the formation of F3- and F4-iPs. Since DHA is extremely rich in the brain, F4-iPs, collectively called neuroprostanes, could be of great importance in assessing the severity of disease states such as Alzheimer's disease. Chemical syntheses of iPF4α-VI and iPF4α-III have been reported.
Recently, another series of isomeric compounds called isofurans has been discovered. These compounds, which contain a substituted tetrahydrofuran (THF) ring, are present at detectable levels in normal tissues and biological fluids. Increased amounts of these compounds have been observed in animal models exposed to high oxidant stress.
The importance of free-radical-generated iPs entails three general areas.
Damage to cell membranes
One consequence of oxidant injury is alteration in the physicochemical properties and function of cellular membranes. This has been attributed to an accumulation of products of lipid peroxidation in lipid bilayers. Phospholipids are essential constituents of the cell membrane. Radical oxygenation of the polyunsaturated fatty acids, located at the sn-2 position of the phospholipids, is bound to create disturbances in the cell membrane. The tightness of the fatty acid portion of the phospholipids is what causes the cell membrane to be hydrophobic and guarantees the integrity of the cell membrane. Polyhydroxylation of the unsaturated fatty acid to yield iPs, which contain three OH groups, is bound to disrupt the tightness of the membrane and have profound effects on the fluidity and integrity of the cellular membrane. For example, a leak could develop in the membrane due to the repulsion by the newly created OH groups and the adjacent lipophilic fatty acids, leading to cell death. See also: Cell membranes
Although the mechanism by which iPs are released from the cell membrane remains to be elucidated, phospholipid-containing iPs are subject to cleavage by phospholipases. These iPs are released in the bloodstream, circulate through the liver and kidney, and eventually are excreted in urine, although their metabolic disposition in vivo remains largely unknown. Several iPs have been shown to have biological effects in vitro. For instance, iPF2α-III is a vasoconstrictor when infused into the rat renal artery and the rabbit pulmonary artery. In both cases the effects were prevented by pharmacological blockage of thromboxane receptors. Similarly, iPF2α-III modulates platelet function. Although it does not cause irreversible aggregation, it facilitates the ability of subthreshold concentrations of conventional agonists to induce this response. These effects require micromolar concentrations of iPF2α-III. Interestingly, much lower concentrations of this compound have been reported to modulate platelet adhesion.
Much less information is available about the potential biological activity of other iPs. For example, iPE2α-III behaves much like iPF2α-III on platelet aggregation. In contrast to these compounds, which appear to act as incidental ligands via the thromboxane receptor, 8,12-iso-iPF2α-III induces proliferative responses in NIH 3T3 cells via the PGF2α receptor. This compound activates this prostanoid receptor selectively and in a saturable manner, but at concentrations roughly one order of magnitude greater than its natural ligand.
Given the difficulty of interpreting the biological potency of individual iPs when multiple species are likely to be formed coincidentally in vivo, it is premature to decide whether the biological properties of these compounds might be relevant under conditions of oxidant stress in vivo. Nonetheless, the possibility of coordinate cellular activation by multiple species, their delivery to cell membranes in highly concentrated form, and the superiority of thromboxane antagonism to cyclooxygenase inhibition in experimental settings suggest a potential role for these compounds as mediators as well as indices of oxidant stress.
Indices of oxidant stress
Perhaps the most interesting aspect of iPs research at present is the possibility that measurement of these compounds in biological fluids might afford a quantitative index of lipid peroxidation in humans. Increased plasma and urinary concentrations of iPF2α-VI and iPF2α-III have been reported in several syndromes thought to be associated with excessive generation of free radicals. These include coronary reperfusion after a period of ischemia, poisoning with paraquat and paracetemol, cigarette smoking–related illnesses, and alcohol-induced liver disease. Isoprostanes, which are increased in low density lipoprotein when it is oxidized in vitro, have been found to be increased in the urine of asymptomatic patients with hyper-cholesterolemia and are present in human atherosclerotic plaque. These observations, and their measurement in urine models of atherosclerosis, raise the likelihood that they may be useful in elucidating the role of LDL and cellular protein oxidation in atherogenesis. In perhaps the most striking evidence to date of the free-radical involvement in atherosclerosis, J. Rokach and G. A. FitzGerald have shown that suppression of elevated iP generation (iPF2α-VI) in vivo reduces the progress of atherosclerosis in apolipoprotein E–deficient mice. Significantly, they have also shown in this study that the level of cholesterol remains high and unchanged. It suggests that in cholesteryl linoleate, the entity present in LDL, the cholesterol moiety is the carrier of the linoleic acid which is responsible for the oxidative stress and the subsequent cell damage. Presumably the cholesterol part is left intact. Recently, the most abundant iP in urine, 8,12-iso-iPF2α-VI (Fig. 4), has been used as an index of oxidant stress and severity of Alzheimer's disease. See also: Cholesterol