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Isoprostanes and phytoprostanes: Bioactive lipids
Biochimie 93 (2011) 52e60
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Isoprostanes and phytoprostanes: Bioactive lipids T. Durand*, V. Bultel-Poncé, A. Guy, S. El Fangour, J.-C. Rossi, J.-M. Galano Institut des Biomolécules Max Mousseron IBMM, UMR 5247 CNRS/Université Montpellier I/Université Montpellier II, Faculté de Pharmacie, 15. Av. Ch. Flahault, F-34093 Montpellier cedex 05, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 November 2009 Accepted 21 May 2010 Available online 2 June 2010
Polyunsaturated fatty acids (PUFA) are important constituents in all eukaryotic organisms, contributing to the structural integrity of biological membranes and serving as precursors for enzymatically-generated local hormones. In addition to these functions, PUFA can generate by a free radical-initiated mechanism, key products which participate in a variety of pathophysiological processes. In particular, free radicalcatalyzed peroxidation of PUFA leads to in vivo formation of isoprostanes (IsoP), neuroprostanes (NeuroP), and phytoprostanes (PhytoP) which display a wide range of biological actions. IsoP are now the most reliable indicators of oxidative stress in humans. In this review, we will discuss some advances in our knowledge regarding two cyclic PUFA derivatives, IsoP and PhytoP, and how their biological roles may be clarified through new approaches based on analytical and synthetic organic chemistry. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Biological activity Polyunsaturated fatty acid Isoprostanes Phytoprostanes Total synthesis
1. Introduction Isoprostanes (IsoP) are generated by the peroxidation of arachidonic acid (AA; C20:4u6) an ubiquitous polyunsaturated fatty acid (PUFA) found esterified to phospholipids in the membranes of animal cells [1]. It is well established that the IsoP possess potent biological activity and are released from tissues in a number of disease states, such as during ischemic injury and neurodegenerative brain disease. In fact, many clinical and basic scientists consider the measurement of IsoP to be the «gold standard» to assess oxidative stress in various human diseases (Fig. 1). Unlike prostaglandins (PG) formed via the action of the cyclooxygenase enzymes, F2-IsoP are formed non-enzymatically as a result of the free radical-mediated peroxidation of AA. While AA and docosahexaenoic acid (DHA; C22:6u3) [2] are abundant in all tissues of mammals, it is noteworthy that brain tissue contains particularly high levels of DHA. By the same peroxidation process, DHA in nerve cell membranes may be converted to neuroprostanes (NeuroP). Of these, research attention has focussed on F4NeuroP which has been identified in vivo and in vitro and appears to be a promising biomarker for various neurodegenerative disorders. In the plant kingdom, PG and IsoP cannot be formed, since higher plants generally lack the enzymatic capacity (chain elongation and desaturase enzymes) to form AA. The predominant PUFAs in plant cell membranes are a-linolenic acid (ALA; C18:3u3) and linoleic acid (C18:2u6) [3]. The free radical-initiated peroxidation of ALA yields compounds termed phytoprostanes (PhytoP) [4]; however, little * Corresponding author. Tel.: þ33 4 67 54 86 22; fax: þ33 4 67 54 86 25. E-mail address: [email protected] (T. Durand). 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.05.014
information is currently available as to what effects PhytoP may have on the function of plant tissues. 2. Toxicity of ROS in lipid peroxidation Oxidative stress and production of reactive oxygen species (ROS) are associated with many pathological processes, including myocardial reperfusion injury and atherosclerosis [5e8]. The leakage of electrons onto O2 during normal metabolism leads to O2 (superoxide anion). However, other sources of O2 exist, notably NADPH oxidase which is responsible for release of superoxide anion from phagocytic cells during the oxidative burst and various metalloenzymes (e.g., cytochrome P450 isoenzymes). Superoxide anion then generates H2O2 (hydrogen peroxide) and OH (hydroxyl radical) via well-characterized mechanisms (Fig. 2). Radicals like OH, but not O2, can target DNA, proteins, and lipids; and one of their major toxic effects is damage to cellular membranes by lipid peroxidation [9]. Non-enzymatic modification of PUFA that contain at least two skipped dienes takes place by a multi-step process when the redox balance of the cell (ROS and O2 concentrations, amount of reducing equivalents in the surrounding tissue), is disturbed and thus the production of initiating species, such as ROS can not be sufficiently suppressed. In early studies, the existence of this condition in a tissue was referred to as “enhanced peroxide tone”. 3. Biosynthesis of cyclic PUFA metabolites The lipid peroxidation process resulting in IsoP formation from AA is presented in Fig. 3. The initial event in the oxidative
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Fig. 1. Peroxidation products of different PUFA: the IsoP, isomers of the prostaglandins, are produced in vivo by a non-enzymatic free radical peroxidation of arachidonic acid initiated by reactive oxygen species. A similar mechanism, also involving hydrogen abstraction, yields NeuroP from docosahexaenoic acid (DHA) and PhytoP from a-linolenic acid (ALA), respectively.
modification of all PUFA, is the abstraction of an hydrogen atom by species such as HO2 (peroxyl radical) or OH. This abstraction can occur at any of the 3 bis-allylic positions, C-7, C-10, C-13; subsequently, the stabilized radical reacts with one molecule of O2 which can lead to two-subsequent five exo-trig cyclizations to generate the cyclopentane ring and allow O2 addition. A final reductive sequence of the endoperoxide and hydroperoxide functionalities leads to four regioisomeric isoP. Since most of the precursor fatty acids are membrane-associated, existing as phospholipid esters in the interior of cell membranes, the formation of IsoP, NeuroP, and PhytoP occurs predominately in esterified form. These cyclic products remain “stored” in the membranes of tissues until they are released by a specific phospholipase. 4. Quantification of cyclic PUFA metabolites In 1990, Morrow et al. [1] showed that a series of prostaglandin F2-like compounds (F2-IsoP) were produced in vivo in humans by a non-cyclooxygenase mechanism involving free radical-catalyzed peroxidation of AA. Levels of these compounds in normal human O2 + H2O2
OH + O2 + OHDNA
Haber-Weiss reaction OH H2O2 + Fe2+ Fenton reaction
Oxidation Non-enzymatic Proteins
OH + Fe3+ + OH -
Lipids
IsoP PhytoP NeuroP
Fig. 2. Reactive oxygen species (ROS) are radicals and intermediates which participate in radical type reactions. The chief route of H2O2 degradation is through reaction with O2 (HabereWeiss reaction). In the presence of Fe2þ, H2O2 can be reduced to the highly reactive OH radical (Fenton reaction). The chain reaction of lipid peroxidation alters the biophysical properties of cellular membranes. Reactive aldehydes and hydroperoxides are formed during the oxidative attack and DNA and proteins can also be targeted.
plasma and urine range from 5 to 40 pg/mL and 500 to 4000 pg/mg of creatinine, respectively. In rats, their formation was found to increase as much as 200-fold in association with marked free radical-catalyzed lipid peroxidation induced by administration of carbon tetrachloride and diquat (Fig. 4). The former is thought to generate a relatively stable trichloromethyl radical whereas the latter is a quinone analogue known to promote redox-cycling and ROS formation. To explore whether these prostanoids exerted biological activity, the effects of one of the compounds formed by this mechanism, 8-epi-prostaglandin F2a (15-F2t-IsoP, IPF2a-III), was examined in rat kidney. Infusion of 8-epi-prostaglandin F2a into a peripheral vein (5 mg/kg per min) or intrarenally (0.5e2.0 mg/kg per min) resulted in marked parallel reductions in renal blood flow and glomerular filtration rate. These results raised the possibility that F2-IsoP might function as pathophysiological mediators in oxidant injury since they exhibited potent biological activity and because their formation was catalyzed by free radicals. Following these groundbreaking experiments, a substantial body of evidence has accumulated indicating that quantification of these compounds is the most accurate, noninvasive method for assessment of oxidant status in humans [10,11]. A number of analytical methodologies have been developed to quantify IsoP [12]. Among them, gas chromatography/ negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS) is the most widely-used approach, and relies on stable isotope dilution [13] for identification and quantification of F2-IsoP, 15-F2t-IsoP, and other F2-IsoP that co-elute with this compound. Several internal standards (IS), deuterated at selected positions, are available from commercial sources to quantify the IsoP. In addition, a number of liquid chromatography e MS/MS methods for F2-IsoP have been developed [14]. Alternative methods have also been developed to quantify IsoP using immunological approaches [15]. A combined LC-immunoassay method may be expected to reduce possible non-specific interference which can be encountered with tissue extracts.
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COOH Arachidonic acid
10 13 ROS attack -H at C10 8 O2 COOH
-H at C13 COOH .
COOH O O .
. O O O
11 .8
COOH O
O 12
[H]
15 O2
O
12 O 2
12
6
COOH .O O
14 11
8
O2
COOH O
. 15 [H]
O
O
COOH O
COOH O
O
O
O
COOH
O OH
COOH
9
O
COOH
O . 8
11
5 .
COOH
O
9
12 O2
O
COOH
O2 5
8
COOH
. 12
[H]
O OH
[H] O OH COOH
O O
O OH
HO COOH HO
7 .
9
10 .
13
O2 11
-H at C7 O2
OH 15-F2-IsoP
HO
OH COOH
HO COOH HO
HO 8-F2-IsoP
OH 12-F2-IsoP
HO
OH COOH
HO 5-F2-IsoP
Fig. 3. IsoP formation from AA: the initial event is the abstraction of a hydrogen atom by species such as O2, OH, or OOH, which can occur at any of the 3 bis-allylic positions, C-7, C-10, C-13. Four regioisomers of IsoP can be produced depending on the site of the initial hydrogen atom abstraction.
In 2008, Mas et al. [16] proposed a new GC-NICI-MS approach to quantify urinary F2-IsoP by using a non-labelled compound as an internal standard: 4(RS)-F4t-NeuroP, which is similar to 15-F2t-IsoP as regards physicochemical properties. The 4(RS)-F4t-NeuroP was synthesized in our laboratory as previously described [17]. Purification of F2-IsoP was achieved by solid-phase extraction, derivatization with pentafluorobenzyl bromide, and subsequent silylation with N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. This method was applied to the determination of F2-IsoP levels in a group of polytraumatized patients (Fig. 5). The urinary level of F2-IsoP measured in controls (healthy volunteers) by this method was 0.811 0.357 ng/mg creatinine, while the level was 4.73 2.70 ng/mg creatinine in the group of polytraumatized patients. These results were in agreement with the results obtained in similar studies [18e20] and clearly demonstrates that F2-IsoP measurement is a valuable analytical tool for monitoring lipid peroxidation in human diseases. Furthermore, our analytical results demonstrate that the use of 4(RS)-F4t-IsoP is a good alternative to a labelled compound which is generally used as IS for quantitative mass spectrometry. 5. Total synthesis of IsoP and PhytoP To systematically examine the biological implications of these PUFA derivatives, the chemical synthesis of pure compounds is needed. Understanding their effects on organ function will be complicated by the fact that these lipids are generated in vivo as mixtures of analogs, although some seem to be formed preferentially. Our efforts have focused on the total synthesis of IsoP and PhytoP. Here, we report our contributions to the field since 1993.
5.1. First generation: original approach to synthesis of IsoP based on radical cyclization Since radical cyclizations are well-suited to form cyclopentane rings with cis-oriented substituents [21], we viewed this as an attractive approach to the synthesis of IsoP intermediates. At the beginning of the nineties, our group along with Rokach’s team, reported the synthesis of a key intermediate, a diastereomer of Corey’s formyl-lactone, via an acyclic thionocarbonate [22]. Subsequently, we developed radical carbocyclizations of functionalized iodo precursors (Scheme 1) [23], which replace the thionocarbonate precursors initially proposed. These precursors were converted to a large set of enantiomerically pure IsoP, NeuroP, and PhytoP by the following standard methods: (a) sequential extension of the side chains by Wittig and/or HWE reactions; (b) protection/deprotection reactions; (c) enantioselective reduction [24,25]. 5.2. Second generation: access to E1-PhytoP and B1-PhytoP Since 2004, our group has concentrated on development of new and flexible routes to B-, D-, E-IsoP and -PhytoP starting from two common intermediates reported by Freimanis, namely the 4-hydroxy-2-cyclopentenone precursors (Scheme 2) [26]. The synthesis started with a Vilsmeier formylation reaction at the fifth position of the furans. A selective rearrangement yielded the 4-hydroxy-2-cyclopentenones after four steps. The two 4hydroxycyclopent-2-enone acetals were transformed to 3-oxocyclopentenecarbaldehydes in three steps and served as precursors for the synthesis of both enantiomers of 16-B1-PhytoP methyl ester as well as of 9-B1-PhytoP methyl ester by Wittig reactions using chiral b-hydroxy phosphonium salts [27].
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5.3. Third generation: a fully flexible approach We have reported recently a simple and highly stereocontrolled strategy toward the total synthesis of isoP based on a bicyclic a,b-epoxy ketone intermediate (Scheme 3). The use of a bicyclo[3.3.0]octene scaffold permitted stereodirection of reagents allowing stereoselective epoxidation, diastereoselective ketone reduction, and regioselective epoxide opening otherwise not attainable with a simple cyclopentene framework [30]. With this process, these two key intermediates (1 in Schemes 3 and 4) can be obtained in 20 g scale without additional chromatographic steps. The total synthesis of 15-F2t-IsoP and its 15 epimer validated this new means of accessing IsoP derivatives from a readily available bicyclo[3.3.0]-octene scaffold; the bicyclic shape allowed the efficient introduction of the four stereocenters of the target compounds through a series of simple synthetic transformations (Scheme 4).
6. IsoP, PhytoP: bioactive lipids in plants and humans
Fig. 4. Effects of administration of diquat (panel A) or carbon tetrachloride (panel B) on plasma levels of F2-prostanoids in rats. Levels of F2-isoprostanes (F2-IsoP) increased as much as 200-fold in association with marked free radical-catalyzed lipid peroxidation induced by administration of CCl4 and diquat. Reprinted with permission from J.L. Roberts II [1].
In another synthetic venture (Scheme 2), methyl trans-3-(2furyl)acrylate was transformed in six steps to racemic 4-hydroxycyclopentenone using the above procedure. An efficient enzymatic resolution for this compound to the two enantiomerically pure hydroxycyclopentenones (R) and (S) using CAL-B was developed [28]. Using this method, we achieved the synthesis of the 15-E2tIsoP and of the 9-E1t-PhytoP [29].
Fig. 5. The method of Mas et al. [16] was employed to measure urinary levels of F2-IsoP in a group of polytraumatized patients. This GC-NICI-MS approach for determination of urinary F2-IsoP uses a non-labelled compound as an internal standard; namely, 4(RS)-F4tNeuroP, which is very similar to 15-F2t-IsoP, in terms of physicochemical properties. The urinary level of F2-IsoP measured in controls by this method was 0.811 0.357 ng/mg creatinine, while the level was 4.73 2.70 ng/mg creatinine in the polytraumatized patients. Reprinted with permission from Elsevier [16].
As pure molecules became available, several studies were conducted in order to determine the significance of these lipids in different areas of biology and medicine. Herein we will discuss some of the outcomes published by a number of coworkers, relating to a possible functional role of these lipids in plants and also linking F2-IsoP biosynthesis to human disease states. Of particular interest, are the convincing studies which point to an immunomodulatory role of these non-enzymatically generated prostanoids in allergic disorders (via the challenge of pollen inhalation) and those studies which have shown the potential value of F2-IsoP as biomarkers of oxidative damage in neurodegenerative and cardiovascular diseases. We will end this survey of their possible clinical relevance, by considering the rationale for dietary supplementation with mono- and polyunsaturated fatty acids, such as the use of triglyceride mixtures which comprise olive or flaxseed oils, in order to prevent cardiovascular disorders.
6.1. Induction of PhytoP antimicrobial metabolites in plants PhytoP have been reported to exhibit a broad spectrum of biological activities in different plant species. The best studied compounds are the cyclopentenone-PhytoP of the A and B classes. Exogenous application of B1-PhytoP regulates gene expression in Arabidopsis thaliana (suspension-cultured cells derived from callus) and tobacco (Nicotiana tabacum cv Xanthi cell suspensions) [31e33]. In these studies, 28% of the up-regulated genes were related to detoxification, stress responses and pathways not associated with chloroplasts or photosynthesis. In agreement with the evidence for enhanced gene expression, the levels of secondary metabolites such as phytoalexins increase upon PhytoP treatment (Fig. 6). It should be emphasized that the stimulatory effect of B1-PhytoP on phytoalexin accumulation was found for multiple classes of phytoalexins in a variety of plant species: the indole-alkaloid camalexin in A. thaliana, the coumarine scopoletin in tobacco, the isobavachalcone in Crotalaria cobalticola and the alkaloid sanguinarine in Eschscholzia californica [32]. Numerous genes involved in the detoxification of xenobiotics were up-regulated by B1-PhytoP, including genes known to respond to structurally diverse compounds in animals; viz., genes controlling the expression of phase I enzymes CytP450 enzymes, aldehyde dehydrogenases, 12-oxo-phytodienoate (OPDA) reductases, phase II conjugating enzymes (glutathione-S-transferases and glycosyltransferases) and putative phase III xenobiotica transporters (ABC transporters) [32].
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Scheme 1. First approach to IsoP synthesis based on radical cyclization according to Durand et al. [23].
6.2. PhytoP prevent cell death in plants The above results suggest that B1-PhytoP may induce adaptative responses by influencing gene expression in plant cells. This kind of adaptive response could provide the plant with a survival advantage, by detoxification of products of lipid peroxidation generated under conditions of oxidative stress (Fig. 7). When Loeffler et al. [32] induced oxidative stress by exposure of tobacco cell suspensions (N. tabacum cv Xanthi cell suspensions) to a low concentration of CuSO4 (75 mM), the number of dead cells increased to 20% above control level (panel A, Fig. 7). By contrast, B1-PhytoP did not influence cell death at the same concentration (panels B and C, Fig. 7). In the same experiments (Fig. 7), very high concentrations of CuSO4 (1 mM or 10 mM) were used to evoke severe oxidant injury, and the effect of preincubation of the cells with B1-PhytoP was examined. The results showed that preincubation of the tobacco cell suspensions with B1-PhytoP (75 mM) significantly decreased cell death. Taken together, these in vitro experiments and the results of the gene expression studies cited above, strongly suggest that B1-PhytoP may be an endogenous
mediator capable of counteracting cell damage caused by various toxicants especially those capable of causing severe oxidative stress [32]. Further studies are needed to determine if B1-PhytoP also exerts a pronounced cytoprotective effect against other transition or active metal cations. 6.3. Signaling lipids: inhibition of IL-12 production in dendritic cells A striking amount of free F1-PhytoP (more than 32,000 mg/g) was found in fresh birch pollen [4]. Moreover, aqueous extracts of Betula alba L. pollen contained E1, F1, A1, and B1-PhytoP, with the amount of E1-PhytoP being eight e fold more abundant than the others [34]. Upon contacting mucosal surfaces, pollen grains do not only release allergens but also can liberate proinflammatory and immunomodulatory lipids, termed pollen-associated lipid mediators (PALM). One of these mediators, the PALM identified as E1-PhytoP, was capable of modulating dendritic cell (DC) function; specifically, E1-PhytoP inhibited in vitro dendritic cell interleukin-12 (IL-12) production and increased Th2 cell polarization. In contrast, in vivo,
Scheme 2. Routes used by Durand’s group for synthesis of B-, D-, E-IsoP and -PhytoP.
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Scheme 3. Multigram-scale preparation of enantiopure precursor Bicyclo[3.3.0]oct-7en-2-ol 1.
both E1-PhytoP and F1-PhytoP caused partial inhibition of cytokine production by Th1 and Th2 cells [35].
inhibitor ozagrel failed to block the effects of these IsoP. Evidence was also presented that 15-A2-IsoP degraded into two biologically active derivatives in vitro, which also inhibited EC tube formation via the TXA2R. Moreover, short hairpin RNAemediated knockdown of the TXA2R antagonized the IsoP-induced effects. In addition, the Rho kinase inhibitor Y-27632 reversed the inhibitory effect of IsoP and the TBXA2 mimetic U-46619 on EC migration and tube formation. It was further demonstrated that the various IsoP exerted a synergistic inhibitory effect on EC tube formation. The study of Benndorf et al. was the first clear demonstration that IsoP inhibit angiogenesis via activation of the TXA2R. By this mechanism, endogenous formation of IsoP may contribute directly to exacerbation of coronary heart disease and to loss of capillary beds in disease states associated with increased oxidative stress.
6.4. Inhibition of angiogenesis by IsoP As stable end products of lipid peroxidation, endogenously formed IsoP are useful markers of oxidative stress and independent risk markers of coronary heart disease. In patients with coronary heart disease, impaired angiogenesis may exacerbate myocardial cell necrosis caused by an insufficient blood supply to the ischemic myocardium. This concept is supported by the work of Benndorf et al. [36] who hypothesized that IsoP may exert detrimental cardiovascular effects by inhibiting angiogenesis. These workers studied the in vitro effects of various IsoP on vascular endothelial growth factor (VEGF)-induced migration and tube formation of human endothelial cells (ECs), and on capillary growth in a cardiac angiogenesis model. In addition, they examined the effect of IsoP on VEGF-induced angiogenesis in the chorioallantoic membrane assay. The latter assay is a widely-used method for evaluating the angiogenic activity of compounds in vivo. A number of the IsoP tested, including 15-F2-, 15-E2-, and 15-A2IsoP; inhibited VEGF-induced migration, tube formation of ECs, and cardiac angiogenesis in vitro, as well as VEGF-induced angiogenesis in vivo. That these effects were dependent on activation of the thromboxane A2 receptor (TXA2R) was demonstrated by pharmacologic blockade with the specific TXA2R antagonists SQ-29548, BM 567, and ICI 192,605. The specific thromboxane A2 synthase
HO
H
S 1
H
1 - LiAlH4, THF -78 °C to rt
O IBX (3 equiv.) DMSO, 90 °C 65% R1O
H
6.5. Is dietary supplementation to elevate endogenous PhytoP beneficial? A tentative answer A recent investigation showed that remarkably high levels of F1PhytoP, E1-PhytoP, A1-PhytoP, and B1-PhytoP are found in vegetable oils and in commercial preparations for parenteral nutrition (Intralipid). The levels of these IsoP were in the range of0.09e99 mg/L [37]. It was also demonstrated that F1-PhytoP was absorbed after oral ingestion, circulated in plasma in a conjugated form, and was excreted in urine as the free acid. Because cyclopentenone-PhytoP display potent anti-inflammatory and apoptosis-inducing activities similar to other prostanoids (e.g., PGA1, deoxy-PGJ2, A2-IsoP, and
tBuOOH, 15 mol% DBU
H
CH2Cl2, 12 h, rt 82%
H
O H CO2H
* R2O
H
HO
2 - Protection 75%
O
H
HO
OH 15-F2t-IsoP
Scheme 4. Durand and Galano’s synthesis of 15-F2t IsoP.
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Fig. 6. Exogenous application of B1-PhytoP regulates gene expression in Arabidopsis thaliana and tobacco. A series of experiments conducted with gene array technology showed that, in these two plant species, 28% of the genes up-regulated in response to B1-PhytoP are related to detoxification, stress responses and secondary metabolism. The accumulation of secondary metabolites following B1-PhytoP treatment was shown for several classes of phytoalexins in different plant species: the coumarine scopoletin in tobacco, the isobavachalcone in Crotalaria cobalticola and the alkaloid sanguinarine in Eschscholzia californica. Reprinted with permission from the American Society of Plant Biologists [32].
Fig. 7. Effect of preincubation with Phytoprostane B1 (PhytoP-B1) enantiomers or a low “priming” concentration of CuSO4 on death of tobacco cells induced by high (millimolar) concentrations of CuSO4. Tobacco cells (Nicotiana tabacum cv Xanthi cell suspensions) were preincubated with a low priming concentration of CuSO4, 75 mM (panel A), 16(S)-PhytoPB1, 75 mM (panel B), or 16(R)-PhytoP-B1, 75 mM (panel C) for 17 h. Thereafter, cells were incubated with 0 mM, 1 mM, or 10 mM CuSO4 for 24 h and the percentage of dead cells was determined. Results obtained after preincubation with CuSO4, 75 mM, or PhytoP-B1 enantiomers are shown as open bars. Results obtained in control preincubations (17 h) with water are shown as filled bars, Dead cells were stained with trypan blue (0.15%, v/v) and counted under the microscope. Reprinted with permission from the American Society of Plant Biologists [32].
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J2-IsoP) this study indicates that PhytoP may contribute to the beneficial effects of the Mediterranean diet. Supplementation with eicosapentaenoic acid (EPA; C20:5u3) and DHA has been reported to reduce lipid peroxidation products (F2-IsoP) formed from AA in healthy humans, as well as in patients with conditions associated with oxidative stress. While the shorter chain PUFA, ALA, is ubiquitous in plant cells and can serve as a precursor to EPA and DHA; its conversion in humans to C20 and C22 metabolites seems to be inefficient. As described above, ALA can also undergo free radical oxidation, forming PhytoP in all plants and leading to the accumulation of high concentrations of PhytoP in plant pollens. In a recent study with healthy male volunteers, Barden et al. [38] examined the effect of ALA supplementation on F1-PhytoP and F2-IsoP concentrations in plasma and urine. The study protocol was as follows: thirty-six non smokers, 20e65 years of age, consumed 9 g/day of either flaxseed oil (62% ALA, 5.4 g/day) or olive oil (placebo) for 4 weeks in a parallel design. At baseline and after 4 weeks of supplementation, blood and a 24-h urine sample were collected for determination of concentrations of F1-PhytoP and F2-IsoP, and selected plasma fatty acids. Compared with the group supplemented with olive oil, the flaxseed oil group showed significantly higher levels of ALA in plasma phospholipids (p < 0.0001), as well as significant elevations of F1-PhytoP in plasma (p ¼ 0.049) and urine (p ¼ 0.06). Flaxseed oil did not affect plasma or urinary F2-IsoP levels. The higher plasma F1-PhytoP concentration in the flaxseed oil group most likely resulted from the increased plasma concentration of the ALA substrate and/or the greater F1-PhytoP content of the flaxseed oil. Future studies are needed to determine the physiological importance of increased plasma and urine F1-PhytoP and their relevance to development of the myocardial infarct and heart disease prevention. 7. Summary and conclusions Our understanding of the role of PUFA peroxidation in the pathogenesis of various human diseases is at an early stage. We know that free radical-induced autoxidation of PUFA occurs in numerous pathological conditions from cardiovascular disorders to cancers and neurodegenerative diseases. Early work with animal tissues indicated that ROS, or radicals formed after exposure to toxicants, are the initial triggers for oxidative injury to membranes. Subsequently, it was shown that IsoP generation accompanied lipid peroxidation injury in mammalian tissues. The exact PUFA derivative generated will depend on the fatty acid composition of the tissue, an important consideration for the use of IsoP or NeuroP as an organ-specific biomarker. Plant cells subjected to oxidative stress also produce cyclopentenone prostanoids, termed PhytoP, by a non-enzymatic mechanism. Through our knowledge of organic chemistry, we can contribute to clinical and basic research by developing novel synthetic approaches and providing samples for biological and analytical work. A number of new approaches for chiral synthesis of IsoP, PhytoP and NeuroP, are now available. Some of these products may be used as markers for the diagnosis and management of patients and will need to be measured accurately and precisely. The contribution of each of these unique PUFA derivatives to tissue and organ damage has to be clearly ascertained within a complex network of signaling molecules and mediators. Acknowledgements We thank our coworkers who are cited in the references. We thank CNRS, the French Ministry of Education and Research, for their continuous support of our research in this field and a part of this work was supported by the University Montpellier I grant
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(BQR-2008). We are also deeply grateful to Pr. Jean-Yves Lallemand and the ICSN for their generous financial support. We thank Dr. Eric G. Spokas (Indian River State College, Fort Pierce, USA) for fruitful discussions, and a critical reading of the manuscript. References [1] J.D. Morrow, K.E. Hill, R.F. Burk, T.M. Nammour, K.F. Badr, L.J. Roberts II, A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc. Natl. Acad. Sci. USA 87 (1990) 9383e9387. [2] L.J. Roberts II, T.J. Montine, W.R. Markesbery, A.R. Tapper, P. Hardy, S. Chemtob, W.D. Dettbarn, J.D. Morrow, Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid. J. Biol. Chem. 273 (1998) 13605e13612. [3] A. Conconi, M. Miquel, J.A. Browse, C.A. Ryan, Intracellular levels of free linolenic and linoleic acid increase in tomato leaves in response to wounding. Plant Physiol. 111 (1996) 797e803. [4] R. Imbusch, M.J. Mueller, Formation of isoprostane F(2)-like compounds (phytoprostanes F(1)) from alpha-linolenic acid in plants. Free Radic. Biol. Med. 28 (2000) 720e726. [5] R. Colavitti, T. Finkel, Reactive oxygen species as mediators of cellular senescence. IUBMB Life 57 (2005) 277e281. [6] D. Il’yasova, A. Ivanova, J.D. Morrow, M. Cesari, M. Pahor, Correlation between two markers of inflammation, serum C-reactive protein and interleukin 6, and indices of oxidative stress in patients with high risk of cardiovascular disease. Biomarkers 13 (2008) 41e51. [7] L.J. Janssen, Isoprostanes and lung vascular pathology. Am. J. Respir. Cell Mol. Biol. 39 (2008) 383e389. [8] D. Steinberg, S. Parthasarathy, T.E. Carew, J.C. Khoo, J.L. Wilztum, Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenecity. N. Engl. J. Med. 320 (1989) 915e924. [9] S. Magder, Reactive oxygen species: toxic molecules or spark of life? Crit. Care 10 (2006) 208e215. [10] G.L. Milne, H. Yin, J.D. Morrow, Human biochemistry of the isoprostane pathway. J. Biol. Chem. 283 (2008) 15533e15537. [11] M.B. Kadiiska, B.C. Gladen, D.D. Baird, D. Germolec, L.B. Graham, C.E. Parker, A. Nyska, J.T. Wachsman, B.N. Ames, S. Basu, N. Brot, G.A. FitzGerald, R.A. Floyd, M. George, J.W. Heinecke, G.E. Hatch, K. Hensley, J.A. Lawson, L.J. Marnett, J.D. Morrow, D.M. Murray, J. Plastaras, L.J. Roberts II, J. Rokach, M.K. Shigenaga, R.S. Sohal, J. Sun, R.R. Tice, D.H. Van Thiel, D. Wellner, P.B. Walter, K.B. Tomer, R.P. Mason, J.C. Barrett, Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning? Free Radic. Biol. Med. 38 (2005) 698e710. [12] O. Berdeaux, O. Scruel, J.L. Cracowski, T. Durand, F2-Isoprostanes: review of analytical methods. Curr. Pharm. Anal. 2 (2006) 69e78. [13] G.L. Milne, H. Yin, J.D. Brooks, S. Sanchez, L.J. Roberts II, J.D. Morrow, Quantification of F2-isoprostanes in biological fluids and tissues as a measure of oxidant stress. Meth. Enzymol. 433 (2007) 113e126. [14] A.W. Taylor, R.S. Bruno, B. Frei, M.G. Traber, Benefits of prolonged gradient separation for high-performance liquid chromatography-tandem mass spectrometry quantitation of plasma total 15-series F2-isoprostanes. Anal. Biochem. 350 (2006) 41e51. [15] S. Basu, Radioimmunoassay of 8-Iso-prostaglandin F2?: an index for oxidative injury via free radical catalyzed lipid peroxidation. Prostaglandins Leukot. Essent. Fatty Acids 58 (1998) 319e325. [16] E. Mas, F. Michel, A. Guy, V. Bultel, Y. Falquet, P. Chardon, J.C. Rossi, J.P. Cristol, T. Durand, Quantification of urinary F2-Isoprostane with 4(RS)-F4t-neuroprostane as an internal standard using gas chromatography-mass spectrometry. J. Chromatogr. B 10 (2008) 5087e5090. [17] T. Durand, A. Guy, J.-P. Vidal, J. Viala, J.-C. Rossi, Total synthesis of 4(RS)-F4tisoprostane methyl ester. Tetrahedron Lett. 41 (2000) 3859e3862. [18] M.F. Walter, J.B. Blumberg, G.G. Dolnikowski, G.J. Handelman, Streamlined F2-isoprostane analysis in plasma and urine with high- performance liquid chromatography and gas chromatography/mass spectroscopy. Anal. Biochem. 280 (2000) 73e79. [19] T.A. Mori, K.D. Croft, I.B. Puddey, L.J. Beilin, An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry. Anal. Biochem. 268 (1999) 117e125. [20] J.D. Morrow, L.J. Roberts II, Mass spectrometry of prostanoids: F2-isoprostanes produced by non-cyclooxygenase free radical-catalyzed mechanism. Methods Enzymol. 233 (1994) 163e174. [21] A.J.J. Beckwith, C.H. Schiesser, Regio- and stereoselectivity of alkenyl radical ring closure: a theoretical study. Tetrahedron 41 (1985) 3925e3941. [22] B. Rondot, T. Durand, J.P. Girard, J.C. Rossi, L. Schio, S.P. Khanapure, J. Rokach, A free radical route to syn lactones and other prostanoid intermediates in isoprostaglandin synthesis. Tetrahedron Lett. 34 (1993) 8245e8248. [23] B. Rondot, T. Durand, J.-C. Rossi, P. Rollin, Synthesis of 3,5-dideoxy-5-iodo-1,2O-isopropylidene-beta-L-lyxo-hexofuranose derivatives. Carbohydr. Res. 261 (1994) 149e156. [24] A. Roland, T. Durand, B. Rondot, J.-P. Vidal, J.-C. Rossi, A practical asymmetric synthesis of ent-12-epi-PGF-2 alpha methyl ester. Bull. Soc. Chim. Fr. 133 (1996) 1149e1154.
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[25] S. El Fangour, A. Guy, V. Despres, J.-P. Vidal, J.-C. Rossi, T. Durand, Total synthesis of the eight diastereomers of the syn-anti-syn phytoprostanes F1 types I and II. J. Org. Chem. 69 (2004) 2498e2503. [26] E. Loza, D. Lola, J. Freimanis, I. Turovskii, M. Gavars, A. Liepina, 2,5-Disubstituted furans in the synthesis of 2-cyclopentenone derivatives. Latvijas PSR Zinatnu Akademijas Vestis, Kimijas Serija 4 (1985) 465e472. [27] S. El Fangour, A. Guy, J.-P. Vidal, J.-C. Rossi, T. Durand, A flexible synthesis of the phytoprostanes B1 type I and II. J. Org. Chem. 70 (2005) 989e997. [28] E. Pinot, A. Guy, A.-L. Guyon, J.-C. Rossi, T. Durand, Enzymatic kinetic resolution of a functionalized 4-hydroxy-cyclopentenone: synthesis of the key intermediates in the total synthesis of isoprostanes. Tetrahedron: Asymmetry 16 (2005) 1893e1895. [29] E. Pinot, A. Guy, A. Fournial, L. Balas, J.-C. Rossi, T. Durand, Total synthesis of the four enantiomerically pure diasteroisomers of the phytoprostanes E1Type II and of the 15-E2t-isoprostanes. J. Org. Chem. 73 (2008) 3063e3069. [30] C. Oger, Y. Brinkmann, S. Bouazzaoui, T. Durand, J.-M. Galano, Stereocontrolled access to isoprostanes via a bicyclo[3.3.0]octene framework. Org. Lett. 10 (2008) 5087e5090. [31] I. Thoma, C. Loeffler, A.K. Sinha, M. Gupta, M. Krischke, B. Steffan, T. Roitsch, M.J. Mueller, Cyclopentenone isoprostanes induced by reactive oxygen species trigger defense gene activation and phytoalexin accumulation in plants. Plant J. 34 (2003) 363e375. [32] C. Loeffler, S. Berger, A. Guy, T. Durand, G. Bringmann, M. Dreyer, U. von Rad, J. Durner, M.J. Mueller, B1-phytoprostanes trigger plant defense and detoxification responses. Plant Physiol. 137 (2005) 328e340.
[33] S. Mueller, B. Hilbert, K. Dueckershoff, T. Roitsch, M. Krischke, M.J. Mueller, S. Berger, General detoxification and stress responses are mediated by oxidized lipids through TGA transcription factors in Arabidopsis. Plant Cell 20 (2008) 768e785. [34] C. Traidl-Hoffmann, V. Mariani, H. Hochrein, K. Karg, H. Wagner, J. Ring, M.J. Mueller, T. Jakob, H. Behrendt, Pollen-associated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization. J. Exp. Med. 201 (2005) 627e636. [35] S. Gilles, V. Mariani, M. Bryce, M.J. Mueller, J. Ring, T. Jakob, S. Pastore, H. Behrendt, C. Traidl-Hoffmann, Pollen-derived E1-phytoprostanes signal via PPAR-{gamma} and NF-{kappa}B-dependent mechanisms. J. Immunol. 182 (2009) 6653e6658. [36] R.A. Benndorf, E. Schwedhelm, A. Gnann, R. Taheri, G. Kom, M. Didie, A. Steenpass, S. Ergun, R.H. Boger, Isoprostanes inhibit vascular endothelial growth factor-induced endothelial cell migration, tube formation, and cardiac vessel sprouting in vitro, as well as angiogenesis in vivo via activation of the thromboxane A2 receptor: a potential link between oxidative stress and impaired angiogenesis. Circ. Res. 103 (2008) 1037e1046. [37] K. Karg, V.M. Dirsch, A.M. Vollmar, J.L. Cracowski, F. Laporte, M.J. Mueller, Biologically active oxidized lipids (phytoprostanes) in the plant diet and parenteral lipid nutrition. Free Radic. Res. 41 (2007) 25e37. [38] A. Barden, K. Croft, T. Durand, A. Guy, M.-J. Mueller, T.J. Mori, F1-phytoprostanes following ALA supplementation in men: a randomised controlled trial. Nutrition 10 (2009) 1890e1895.
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Mini-review
Isoprostanes and phytoprostanes: Bioactive lipids T. Durand*, V. Bultel-Poncé, A. Guy, S. El Fangour, J.-C. Rossi, J.-M. Galano Institut des Biomolécules Max Mousseron IBMM, UMR 5247 CNRS/Université Montpellier I/Université Montpellier II, Faculté de Pharmacie, 15. Av. Ch. Flahault, F-34093 Montpellier cedex 05, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 November 2009 Accepted 21 May 2010 Available online 2 June 2010
Polyunsaturated fatty acids (PUFA) are important constituents in all eukaryotic organisms, contributing to the structural integrity of biological membranes and serving as precursors for enzymatically-generated local hormones. In addition to these functions, PUFA can generate by a free radical-initiated mechanism, key products which participate in a variety of pathophysiological processes. In particular, free radicalcatalyzed peroxidation of PUFA leads to in vivo formation of isoprostanes (IsoP), neuroprostanes (NeuroP), and phytoprostanes (PhytoP) which display a wide range of biological actions. IsoP are now the most reliable indicators of oxidative stress in humans. In this review, we will discuss some advances in our knowledge regarding two cyclic PUFA derivatives, IsoP and PhytoP, and how their biological roles may be clarified through new approaches based on analytical and synthetic organic chemistry. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Biological activity Polyunsaturated fatty acid Isoprostanes Phytoprostanes Total synthesis
1. Introduction Isoprostanes (IsoP) are generated by the peroxidation of arachidonic acid (AA; C20:4u6) an ubiquitous polyunsaturated fatty acid (PUFA) found esterified to phospholipids in the membranes of animal cells [1]. It is well established that the IsoP possess potent biological activity and are released from tissues in a number of disease states, such as during ischemic injury and neurodegenerative brain disease. In fact, many clinical and basic scientists consider the measurement of IsoP to be the «gold standard» to assess oxidative stress in various human diseases (Fig. 1). Unlike prostaglandins (PG) formed via the action of the cyclooxygenase enzymes, F2-IsoP are formed non-enzymatically as a result of the free radical-mediated peroxidation of AA. While AA and docosahexaenoic acid (DHA; C22:6u3) [2] are abundant in all tissues of mammals, it is noteworthy that brain tissue contains particularly high levels of DHA. By the same peroxidation process, DHA in nerve cell membranes may be converted to neuroprostanes (NeuroP). Of these, research attention has focussed on F4NeuroP which has been identified in vivo and in vitro and appears to be a promising biomarker for various neurodegenerative disorders. In the plant kingdom, PG and IsoP cannot be formed, since higher plants generally lack the enzymatic capacity (chain elongation and desaturase enzymes) to form AA. The predominant PUFAs in plant cell membranes are a-linolenic acid (ALA; C18:3u3) and linoleic acid (C18:2u6) [3]. The free radical-initiated peroxidation of ALA yields compounds termed phytoprostanes (PhytoP) [4]; however, little * Corresponding author. Tel.: þ33 4 67 54 86 22; fax: þ33 4 67 54 86 25. E-mail address: [email protected] (T. Durand). 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.05.014
information is currently available as to what effects PhytoP may have on the function of plant tissues. 2. Toxicity of ROS in lipid peroxidation Oxidative stress and production of reactive oxygen species (ROS) are associated with many pathological processes, including myocardial reperfusion injury and atherosclerosis [5e8]. The leakage of electrons onto O2 during normal metabolism leads to O2 (superoxide anion). However, other sources of O2 exist, notably NADPH oxidase which is responsible for release of superoxide anion from phagocytic cells during the oxidative burst and various metalloenzymes (e.g., cytochrome P450 isoenzymes). Superoxide anion then generates H2O2 (hydrogen peroxide) and OH (hydroxyl radical) via well-characterized mechanisms (Fig. 2). Radicals like OH, but not O2, can target DNA, proteins, and lipids; and one of their major toxic effects is damage to cellular membranes by lipid peroxidation [9]. Non-enzymatic modification of PUFA that contain at least two skipped dienes takes place by a multi-step process when the redox balance of the cell (ROS and O2 concentrations, amount of reducing equivalents in the surrounding tissue), is disturbed and thus the production of initiating species, such as ROS can not be sufficiently suppressed. In early studies, the existence of this condition in a tissue was referred to as “enhanced peroxide tone”. 3. Biosynthesis of cyclic PUFA metabolites The lipid peroxidation process resulting in IsoP formation from AA is presented in Fig. 3. The initial event in the oxidative
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Fig. 1. Peroxidation products of different PUFA: the IsoP, isomers of the prostaglandins, are produced in vivo by a non-enzymatic free radical peroxidation of arachidonic acid initiated by reactive oxygen species. A similar mechanism, also involving hydrogen abstraction, yields NeuroP from docosahexaenoic acid (DHA) and PhytoP from a-linolenic acid (ALA), respectively.
modification of all PUFA, is the abstraction of an hydrogen atom by species such as HO2 (peroxyl radical) or OH. This abstraction can occur at any of the 3 bis-allylic positions, C-7, C-10, C-13; subsequently, the stabilized radical reacts with one molecule of O2 which can lead to two-subsequent five exo-trig cyclizations to generate the cyclopentane ring and allow O2 addition. A final reductive sequence of the endoperoxide and hydroperoxide functionalities leads to four regioisomeric isoP. Since most of the precursor fatty acids are membrane-associated, existing as phospholipid esters in the interior of cell membranes, the formation of IsoP, NeuroP, and PhytoP occurs predominately in esterified form. These cyclic products remain “stored” in the membranes of tissues until they are released by a specific phospholipase. 4. Quantification of cyclic PUFA metabolites In 1990, Morrow et al. [1] showed that a series of prostaglandin F2-like compounds (F2-IsoP) were produced in vivo in humans by a non-cyclooxygenase mechanism involving free radical-catalyzed peroxidation of AA. Levels of these compounds in normal human O2 + H2O2
OH + O2 + OHDNA
Haber-Weiss reaction OH H2O2 + Fe2+ Fenton reaction
Oxidation Non-enzymatic Proteins
OH + Fe3+ + OH -
Lipids
IsoP PhytoP NeuroP
Fig. 2. Reactive oxygen species (ROS) are radicals and intermediates which participate in radical type reactions. The chief route of H2O2 degradation is through reaction with O2 (HabereWeiss reaction). In the presence of Fe2þ, H2O2 can be reduced to the highly reactive OH radical (Fenton reaction). The chain reaction of lipid peroxidation alters the biophysical properties of cellular membranes. Reactive aldehydes and hydroperoxides are formed during the oxidative attack and DNA and proteins can also be targeted.
plasma and urine range from 5 to 40 pg/mL and 500 to 4000 pg/mg of creatinine, respectively. In rats, their formation was found to increase as much as 200-fold in association with marked free radical-catalyzed lipid peroxidation induced by administration of carbon tetrachloride and diquat (Fig. 4). The former is thought to generate a relatively stable trichloromethyl radical whereas the latter is a quinone analogue known to promote redox-cycling and ROS formation. To explore whether these prostanoids exerted biological activity, the effects of one of the compounds formed by this mechanism, 8-epi-prostaglandin F2a (15-F2t-IsoP, IPF2a-III), was examined in rat kidney. Infusion of 8-epi-prostaglandin F2a into a peripheral vein (5 mg/kg per min) or intrarenally (0.5e2.0 mg/kg per min) resulted in marked parallel reductions in renal blood flow and glomerular filtration rate. These results raised the possibility that F2-IsoP might function as pathophysiological mediators in oxidant injury since they exhibited potent biological activity and because their formation was catalyzed by free radicals. Following these groundbreaking experiments, a substantial body of evidence has accumulated indicating that quantification of these compounds is the most accurate, noninvasive method for assessment of oxidant status in humans [10,11]. A number of analytical methodologies have been developed to quantify IsoP [12]. Among them, gas chromatography/ negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS) is the most widely-used approach, and relies on stable isotope dilution [13] for identification and quantification of F2-IsoP, 15-F2t-IsoP, and other F2-IsoP that co-elute with this compound. Several internal standards (IS), deuterated at selected positions, are available from commercial sources to quantify the IsoP. In addition, a number of liquid chromatography e MS/MS methods for F2-IsoP have been developed [14]. Alternative methods have also been developed to quantify IsoP using immunological approaches [15]. A combined LC-immunoassay method may be expected to reduce possible non-specific interference which can be encountered with tissue extracts.
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7
COOH Arachidonic acid
10 13 ROS attack -H at C10 8 O2 COOH
-H at C13 COOH .
COOH O O .
. O O O
11 .8
COOH O
O 12
[H]
15 O2
O
12 O 2
12
6
COOH .O O
14 11
8
O2
COOH O
. 15 [H]
O
O
COOH O
COOH O
O
O
O
COOH
O OH
COOH
9
O
COOH
O . 8
11
5 .
COOH
O
9
12 O2
O
COOH
O2 5
8
COOH
. 12
[H]
O OH
[H] O OH COOH
O O
O OH
HO COOH HO
7 .
9
10 .
13
O2 11
-H at C7 O2
OH 15-F2-IsoP
HO
OH COOH
HO COOH HO
HO 8-F2-IsoP
OH 12-F2-IsoP
HO
OH COOH
HO 5-F2-IsoP
Fig. 3. IsoP formation from AA: the initial event is the abstraction of a hydrogen atom by species such as O2, OH, or OOH, which can occur at any of the 3 bis-allylic positions, C-7, C-10, C-13. Four regioisomers of IsoP can be produced depending on the site of the initial hydrogen atom abstraction.
In 2008, Mas et al. [16] proposed a new GC-NICI-MS approach to quantify urinary F2-IsoP by using a non-labelled compound as an internal standard: 4(RS)-F4t-NeuroP, which is similar to 15-F2t-IsoP as regards physicochemical properties. The 4(RS)-F4t-NeuroP was synthesized in our laboratory as previously described [17]. Purification of F2-IsoP was achieved by solid-phase extraction, derivatization with pentafluorobenzyl bromide, and subsequent silylation with N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. This method was applied to the determination of F2-IsoP levels in a group of polytraumatized patients (Fig. 5). The urinary level of F2-IsoP measured in controls (healthy volunteers) by this method was 0.811 0.357 ng/mg creatinine, while the level was 4.73 2.70 ng/mg creatinine in the group of polytraumatized patients. These results were in agreement with the results obtained in similar studies [18e20] and clearly demonstrates that F2-IsoP measurement is a valuable analytical tool for monitoring lipid peroxidation in human diseases. Furthermore, our analytical results demonstrate that the use of 4(RS)-F4t-IsoP is a good alternative to a labelled compound which is generally used as IS for quantitative mass spectrometry. 5. Total synthesis of IsoP and PhytoP To systematically examine the biological implications of these PUFA derivatives, the chemical synthesis of pure compounds is needed. Understanding their effects on organ function will be complicated by the fact that these lipids are generated in vivo as mixtures of analogs, although some seem to be formed preferentially. Our efforts have focused on the total synthesis of IsoP and PhytoP. Here, we report our contributions to the field since 1993.
5.1. First generation: original approach to synthesis of IsoP based on radical cyclization Since radical cyclizations are well-suited to form cyclopentane rings with cis-oriented substituents [21], we viewed this as an attractive approach to the synthesis of IsoP intermediates. At the beginning of the nineties, our group along with Rokach’s team, reported the synthesis of a key intermediate, a diastereomer of Corey’s formyl-lactone, via an acyclic thionocarbonate [22]. Subsequently, we developed radical carbocyclizations of functionalized iodo precursors (Scheme 1) [23], which replace the thionocarbonate precursors initially proposed. These precursors were converted to a large set of enantiomerically pure IsoP, NeuroP, and PhytoP by the following standard methods: (a) sequential extension of the side chains by Wittig and/or HWE reactions; (b) protection/deprotection reactions; (c) enantioselective reduction [24,25]. 5.2. Second generation: access to E1-PhytoP and B1-PhytoP Since 2004, our group has concentrated on development of new and flexible routes to B-, D-, E-IsoP and -PhytoP starting from two common intermediates reported by Freimanis, namely the 4-hydroxy-2-cyclopentenone precursors (Scheme 2) [26]. The synthesis started with a Vilsmeier formylation reaction at the fifth position of the furans. A selective rearrangement yielded the 4-hydroxy-2-cyclopentenones after four steps. The two 4hydroxycyclopent-2-enone acetals were transformed to 3-oxocyclopentenecarbaldehydes in three steps and served as precursors for the synthesis of both enantiomers of 16-B1-PhytoP methyl ester as well as of 9-B1-PhytoP methyl ester by Wittig reactions using chiral b-hydroxy phosphonium salts [27].
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5.3. Third generation: a fully flexible approach We have reported recently a simple and highly stereocontrolled strategy toward the total synthesis of isoP based on a bicyclic a,b-epoxy ketone intermediate (Scheme 3). The use of a bicyclo[3.3.0]octene scaffold permitted stereodirection of reagents allowing stereoselective epoxidation, diastereoselective ketone reduction, and regioselective epoxide opening otherwise not attainable with a simple cyclopentene framework [30]. With this process, these two key intermediates (1 in Schemes 3 and 4) can be obtained in 20 g scale without additional chromatographic steps. The total synthesis of 15-F2t-IsoP and its 15 epimer validated this new means of accessing IsoP derivatives from a readily available bicyclo[3.3.0]-octene scaffold; the bicyclic shape allowed the efficient introduction of the four stereocenters of the target compounds through a series of simple synthetic transformations (Scheme 4).
6. IsoP, PhytoP: bioactive lipids in plants and humans
Fig. 4. Effects of administration of diquat (panel A) or carbon tetrachloride (panel B) on plasma levels of F2-prostanoids in rats. Levels of F2-isoprostanes (F2-IsoP) increased as much as 200-fold in association with marked free radical-catalyzed lipid peroxidation induced by administration of CCl4 and diquat. Reprinted with permission from J.L. Roberts II [1].
In another synthetic venture (Scheme 2), methyl trans-3-(2furyl)acrylate was transformed in six steps to racemic 4-hydroxycyclopentenone using the above procedure. An efficient enzymatic resolution for this compound to the two enantiomerically pure hydroxycyclopentenones (R) and (S) using CAL-B was developed [28]. Using this method, we achieved the synthesis of the 15-E2tIsoP and of the 9-E1t-PhytoP [29].
Fig. 5. The method of Mas et al. [16] was employed to measure urinary levels of F2-IsoP in a group of polytraumatized patients. This GC-NICI-MS approach for determination of urinary F2-IsoP uses a non-labelled compound as an internal standard; namely, 4(RS)-F4tNeuroP, which is very similar to 15-F2t-IsoP, in terms of physicochemical properties. The urinary level of F2-IsoP measured in controls by this method was 0.811 0.357 ng/mg creatinine, while the level was 4.73 2.70 ng/mg creatinine in the polytraumatized patients. Reprinted with permission from Elsevier [16].
As pure molecules became available, several studies were conducted in order to determine the significance of these lipids in different areas of biology and medicine. Herein we will discuss some of the outcomes published by a number of coworkers, relating to a possible functional role of these lipids in plants and also linking F2-IsoP biosynthesis to human disease states. Of particular interest, are the convincing studies which point to an immunomodulatory role of these non-enzymatically generated prostanoids in allergic disorders (via the challenge of pollen inhalation) and those studies which have shown the potential value of F2-IsoP as biomarkers of oxidative damage in neurodegenerative and cardiovascular diseases. We will end this survey of their possible clinical relevance, by considering the rationale for dietary supplementation with mono- and polyunsaturated fatty acids, such as the use of triglyceride mixtures which comprise olive or flaxseed oils, in order to prevent cardiovascular disorders.
6.1. Induction of PhytoP antimicrobial metabolites in plants PhytoP have been reported to exhibit a broad spectrum of biological activities in different plant species. The best studied compounds are the cyclopentenone-PhytoP of the A and B classes. Exogenous application of B1-PhytoP regulates gene expression in Arabidopsis thaliana (suspension-cultured cells derived from callus) and tobacco (Nicotiana tabacum cv Xanthi cell suspensions) [31e33]. In these studies, 28% of the up-regulated genes were related to detoxification, stress responses and pathways not associated with chloroplasts or photosynthesis. In agreement with the evidence for enhanced gene expression, the levels of secondary metabolites such as phytoalexins increase upon PhytoP treatment (Fig. 6). It should be emphasized that the stimulatory effect of B1-PhytoP on phytoalexin accumulation was found for multiple classes of phytoalexins in a variety of plant species: the indole-alkaloid camalexin in A. thaliana, the coumarine scopoletin in tobacco, the isobavachalcone in Crotalaria cobalticola and the alkaloid sanguinarine in Eschscholzia californica [32]. Numerous genes involved in the detoxification of xenobiotics were up-regulated by B1-PhytoP, including genes known to respond to structurally diverse compounds in animals; viz., genes controlling the expression of phase I enzymes CytP450 enzymes, aldehyde dehydrogenases, 12-oxo-phytodienoate (OPDA) reductases, phase II conjugating enzymes (glutathione-S-transferases and glycosyltransferases) and putative phase III xenobiotica transporters (ABC transporters) [32].
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Scheme 1. First approach to IsoP synthesis based on radical cyclization according to Durand et al. [23].
6.2. PhytoP prevent cell death in plants The above results suggest that B1-PhytoP may induce adaptative responses by influencing gene expression in plant cells. This kind of adaptive response could provide the plant with a survival advantage, by detoxification of products of lipid peroxidation generated under conditions of oxidative stress (Fig. 7). When Loeffler et al. [32] induced oxidative stress by exposure of tobacco cell suspensions (N. tabacum cv Xanthi cell suspensions) to a low concentration of CuSO4 (75 mM), the number of dead cells increased to 20% above control level (panel A, Fig. 7). By contrast, B1-PhytoP did not influence cell death at the same concentration (panels B and C, Fig. 7). In the same experiments (Fig. 7), very high concentrations of CuSO4 (1 mM or 10 mM) were used to evoke severe oxidant injury, and the effect of preincubation of the cells with B1-PhytoP was examined. The results showed that preincubation of the tobacco cell suspensions with B1-PhytoP (75 mM) significantly decreased cell death. Taken together, these in vitro experiments and the results of the gene expression studies cited above, strongly suggest that B1-PhytoP may be an endogenous
mediator capable of counteracting cell damage caused by various toxicants especially those capable of causing severe oxidative stress [32]. Further studies are needed to determine if B1-PhytoP also exerts a pronounced cytoprotective effect against other transition or active metal cations. 6.3. Signaling lipids: inhibition of IL-12 production in dendritic cells A striking amount of free F1-PhytoP (more than 32,000 mg/g) was found in fresh birch pollen [4]. Moreover, aqueous extracts of Betula alba L. pollen contained E1, F1, A1, and B1-PhytoP, with the amount of E1-PhytoP being eight e fold more abundant than the others [34]. Upon contacting mucosal surfaces, pollen grains do not only release allergens but also can liberate proinflammatory and immunomodulatory lipids, termed pollen-associated lipid mediators (PALM). One of these mediators, the PALM identified as E1-PhytoP, was capable of modulating dendritic cell (DC) function; specifically, E1-PhytoP inhibited in vitro dendritic cell interleukin-12 (IL-12) production and increased Th2 cell polarization. In contrast, in vivo,
Scheme 2. Routes used by Durand’s group for synthesis of B-, D-, E-IsoP and -PhytoP.
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Scheme 3. Multigram-scale preparation of enantiopure precursor Bicyclo[3.3.0]oct-7en-2-ol 1.
both E1-PhytoP and F1-PhytoP caused partial inhibition of cytokine production by Th1 and Th2 cells [35].
inhibitor ozagrel failed to block the effects of these IsoP. Evidence was also presented that 15-A2-IsoP degraded into two biologically active derivatives in vitro, which also inhibited EC tube formation via the TXA2R. Moreover, short hairpin RNAemediated knockdown of the TXA2R antagonized the IsoP-induced effects. In addition, the Rho kinase inhibitor Y-27632 reversed the inhibitory effect of IsoP and the TBXA2 mimetic U-46619 on EC migration and tube formation. It was further demonstrated that the various IsoP exerted a synergistic inhibitory effect on EC tube formation. The study of Benndorf et al. was the first clear demonstration that IsoP inhibit angiogenesis via activation of the TXA2R. By this mechanism, endogenous formation of IsoP may contribute directly to exacerbation of coronary heart disease and to loss of capillary beds in disease states associated with increased oxidative stress.
6.4. Inhibition of angiogenesis by IsoP As stable end products of lipid peroxidation, endogenously formed IsoP are useful markers of oxidative stress and independent risk markers of coronary heart disease. In patients with coronary heart disease, impaired angiogenesis may exacerbate myocardial cell necrosis caused by an insufficient blood supply to the ischemic myocardium. This concept is supported by the work of Benndorf et al. [36] who hypothesized that IsoP may exert detrimental cardiovascular effects by inhibiting angiogenesis. These workers studied the in vitro effects of various IsoP on vascular endothelial growth factor (VEGF)-induced migration and tube formation of human endothelial cells (ECs), and on capillary growth in a cardiac angiogenesis model. In addition, they examined the effect of IsoP on VEGF-induced angiogenesis in the chorioallantoic membrane assay. The latter assay is a widely-used method for evaluating the angiogenic activity of compounds in vivo. A number of the IsoP tested, including 15-F2-, 15-E2-, and 15-A2IsoP; inhibited VEGF-induced migration, tube formation of ECs, and cardiac angiogenesis in vitro, as well as VEGF-induced angiogenesis in vivo. That these effects were dependent on activation of the thromboxane A2 receptor (TXA2R) was demonstrated by pharmacologic blockade with the specific TXA2R antagonists SQ-29548, BM 567, and ICI 192,605. The specific thromboxane A2 synthase
HO
H
S 1
H
1 - LiAlH4, THF -78 °C to rt
O IBX (3 equiv.) DMSO, 90 °C 65% R1O
H
6.5. Is dietary supplementation to elevate endogenous PhytoP beneficial? A tentative answer A recent investigation showed that remarkably high levels of F1PhytoP, E1-PhytoP, A1-PhytoP, and B1-PhytoP are found in vegetable oils and in commercial preparations for parenteral nutrition (Intralipid). The levels of these IsoP were in the range of0.09e99 mg/L [37]. It was also demonstrated that F1-PhytoP was absorbed after oral ingestion, circulated in plasma in a conjugated form, and was excreted in urine as the free acid. Because cyclopentenone-PhytoP display potent anti-inflammatory and apoptosis-inducing activities similar to other prostanoids (e.g., PGA1, deoxy-PGJ2, A2-IsoP, and
tBuOOH, 15 mol% DBU
H
CH2Cl2, 12 h, rt 82%
H
O H CO2H
* R2O
H
HO
2 - Protection 75%
O
H
HO
OH 15-F2t-IsoP
Scheme 4. Durand and Galano’s synthesis of 15-F2t IsoP.
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T. Durand et al. / Biochimie 93 (2011) 52e60
Fig. 6. Exogenous application of B1-PhytoP regulates gene expression in Arabidopsis thaliana and tobacco. A series of experiments conducted with gene array technology showed that, in these two plant species, 28% of the genes up-regulated in response to B1-PhytoP are related to detoxification, stress responses and secondary metabolism. The accumulation of secondary metabolites following B1-PhytoP treatment was shown for several classes of phytoalexins in different plant species: the coumarine scopoletin in tobacco, the isobavachalcone in Crotalaria cobalticola and the alkaloid sanguinarine in Eschscholzia californica. Reprinted with permission from the American Society of Plant Biologists [32].
Fig. 7. Effect of preincubation with Phytoprostane B1 (PhytoP-B1) enantiomers or a low “priming” concentration of CuSO4 on death of tobacco cells induced by high (millimolar) concentrations of CuSO4. Tobacco cells (Nicotiana tabacum cv Xanthi cell suspensions) were preincubated with a low priming concentration of CuSO4, 75 mM (panel A), 16(S)-PhytoPB1, 75 mM (panel B), or 16(R)-PhytoP-B1, 75 mM (panel C) for 17 h. Thereafter, cells were incubated with 0 mM, 1 mM, or 10 mM CuSO4 for 24 h and the percentage of dead cells was determined. Results obtained after preincubation with CuSO4, 75 mM, or PhytoP-B1 enantiomers are shown as open bars. Results obtained in control preincubations (17 h) with water are shown as filled bars, Dead cells were stained with trypan blue (0.15%, v/v) and counted under the microscope. Reprinted with permission from the American Society of Plant Biologists [32].
T. Durand et al. / Biochimie 93 (2011) 52e60
J2-IsoP) this study indicates that PhytoP may contribute to the beneficial effects of the Mediterranean diet. Supplementation with eicosapentaenoic acid (EPA; C20:5u3) and DHA has been reported to reduce lipid peroxidation products (F2-IsoP) formed from AA in healthy humans, as well as in patients with conditions associated with oxidative stress. While the shorter chain PUFA, ALA, is ubiquitous in plant cells and can serve as a precursor to EPA and DHA; its conversion in humans to C20 and C22 metabolites seems to be inefficient. As described above, ALA can also undergo free radical oxidation, forming PhytoP in all plants and leading to the accumulation of high concentrations of PhytoP in plant pollens. In a recent study with healthy male volunteers, Barden et al. [38] examined the effect of ALA supplementation on F1-PhytoP and F2-IsoP concentrations in plasma and urine. The study protocol was as follows: thirty-six non smokers, 20e65 years of age, consumed 9 g/day of either flaxseed oil (62% ALA, 5.4 g/day) or olive oil (placebo) for 4 weeks in a parallel design. At baseline and after 4 weeks of supplementation, blood and a 24-h urine sample were collected for determination of concentrations of F1-PhytoP and F2-IsoP, and selected plasma fatty acids. Compared with the group supplemented with olive oil, the flaxseed oil group showed significantly higher levels of ALA in plasma phospholipids (p < 0.0001), as well as significant elevations of F1-PhytoP in plasma (p ¼ 0.049) and urine (p ¼ 0.06). Flaxseed oil did not affect plasma or urinary F2-IsoP levels. The higher plasma F1-PhytoP concentration in the flaxseed oil group most likely resulted from the increased plasma concentration of the ALA substrate and/or the greater F1-PhytoP content of the flaxseed oil. Future studies are needed to determine the physiological importance of increased plasma and urine F1-PhytoP and their relevance to development of the myocardial infarct and heart disease prevention. 7. Summary and conclusions Our understanding of the role of PUFA peroxidation in the pathogenesis of various human diseases is at an early stage. We know that free radical-induced autoxidation of PUFA occurs in numerous pathological conditions from cardiovascular disorders to cancers and neurodegenerative diseases. Early work with animal tissues indicated that ROS, or radicals formed after exposure to toxicants, are the initial triggers for oxidative injury to membranes. Subsequently, it was shown that IsoP generation accompanied lipid peroxidation injury in mammalian tissues. The exact PUFA derivative generated will depend on the fatty acid composition of the tissue, an important consideration for the use of IsoP or NeuroP as an organ-specific biomarker. Plant cells subjected to oxidative stress also produce cyclopentenone prostanoids, termed PhytoP, by a non-enzymatic mechanism. Through our knowledge of organic chemistry, we can contribute to clinical and basic research by developing novel synthetic approaches and providing samples for biological and analytical work. A number of new approaches for chiral synthesis of IsoP, PhytoP and NeuroP, are now available. Some of these products may be used as markers for the diagnosis and management of patients and will need to be measured accurately and precisely. The contribution of each of these unique PUFA derivatives to tissue and organ damage has to be clearly ascertained within a complex network of signaling molecules and mediators. Acknowledgements We thank our coworkers who are cited in the references. We thank CNRS, the French Ministry of Education and Research, for their continuous support of our research in this field and a part of this work was supported by the University Montpellier I grant
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(BQR-2008). We are also deeply grateful to Pr. Jean-Yves Lallemand and the ICSN for their generous financial support. We thank Dr. Eric G. Spokas (Indian River State College, Fort Pierce, USA) for fruitful discussions, and a critical reading of the manuscript. References [1] J.D. Morrow, K.E. Hill, R.F. Burk, T.M. Nammour, K.F. Badr, L.J. Roberts II, A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc. Natl. Acad. Sci. USA 87 (1990) 9383e9387. [2] L.J. Roberts II, T.J. Montine, W.R. Markesbery, A.R. Tapper, P. Hardy, S. Chemtob, W.D. Dettbarn, J.D. Morrow, Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid. J. Biol. Chem. 273 (1998) 13605e13612. [3] A. Conconi, M. Miquel, J.A. Browse, C.A. Ryan, Intracellular levels of free linolenic and linoleic acid increase in tomato leaves in response to wounding. Plant Physiol. 111 (1996) 797e803. [4] R. Imbusch, M.J. 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[33] S. Mueller, B. Hilbert, K. Dueckershoff, T. Roitsch, M. Krischke, M.J. Mueller, S. Berger, General detoxification and stress responses are mediated by oxidized lipids through TGA transcription factors in Arabidopsis. Plant Cell 20 (2008) 768e785. [34] C. Traidl-Hoffmann, V. Mariani, H. Hochrein, K. Karg, H. Wagner, J. Ring, M.J. Mueller, T. Jakob, H. Behrendt, Pollen-associated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization. J. Exp. Med. 201 (2005) 627e636. [35] S. Gilles, V. Mariani, M. Bryce, M.J. Mueller, J. Ring, T. Jakob, S. Pastore, H. Behrendt, C. Traidl-Hoffmann, Pollen-derived E1-phytoprostanes signal via PPAR-{gamma} and NF-{kappa}B-dependent mechanisms. J. Immunol. 182 (2009) 6653e6658. [36] R.A. Benndorf, E. Schwedhelm, A. Gnann, R. Taheri, G. Kom, M. Didie, A. Steenpass, S. Ergun, R.H. Boger, Isoprostanes inhibit vascular endothelial growth factor-induced endothelial cell migration, tube formation, and cardiac vessel sprouting in vitro, as well as angiogenesis in vivo via activation of the thromboxane A2 receptor: a potential link between oxidative stress and impaired angiogenesis. Circ. Res. 103 (2008) 1037e1046. [37] K. Karg, V.M. Dirsch, A.M. Vollmar, J.L. Cracowski, F. Laporte, M.J. Mueller, Biologically active oxidized lipids (phytoprostanes) in the plant diet and parenteral lipid nutrition. Free Radic. Res. 41 (2007) 25e37. [38] A. Barden, K. Croft, T. Durand, A. Guy, M.-J. Mueller, T.J. Mori, F1-phytoprostanes following ALA supplementation in men: a randomised controlled trial. Nutrition 10 (2009) 1890e1895.