MX2008001391A - Oxidised phospholipids comprising a fluorophore moiety and use in the determination of the presence of enzymes having antiatherogenetic activity - Google Patents

Abstract

The invention relates to oxidized phospholipids having one of the general formulas (I) or (II) wherein A=O, C, NH, or S;B=O, C, NH, or S;and R2is selected from the group consisting of -CO-(CH2)n-CH3;-CO-(CH2)n-CHO;and -CO-(CH2)n-COOH, with n=3-7, with the proviso that in general formula (I), R1is selected from the group consisting of -CH2-(CH2)n-X;and -CO-(CH2)n-X with n=5-11, wherein X is a fluorophore;and in general formula (II), R1is selected from the group consisting of -CH=CH-(CH2)n-CH3with n=9-15;-(CH2)n-CH3with n=11-17;and -CO-(CH2)n-CH3with n - 10-16;and R3is selected from the group consisting of -CO-(CH2)n-X;and -SO2-(CH2)n-X, with n=0-5, wherein X is a fluorophore.

Description

OXIDATED PHOSPHOLIPIDES THAT COMPRISE A FLUOROFORA PORTION AND USE IN DETERMINING THE PRESENCE OF ENZYMES THAT HAVE ANTIATEROGENETIC ACTIVITY DESCRIPTIVE MEMORY Oxidative modification of LDL is considered an important factor in atherosclerosis. Studies from several laboratories have revealed that the biological effects triggered by mmLDL can be attributed largely to the phospholipid oxidation products (Leitinger, N et al (1999) PNAS 96, 12010-12015; Watson, AD et al. 1995) J. Clin, Invest 95, 774-782, Leitinger, N. et al (1997) Adv. Exp. Med. Biol. 433, 379-382; LoidI, A. et al. (2003) J. Biol. Chem. 278, 32921-32928). Their highest levels in atherosclerotic plaques (Watson, AD et al (1997) J. Biol. Chem. 272, 13597-13607; Itabe, H. et al. (1997) J. Biol. Chem. 269, 15274-15279) and high titers of antibody against oxidized phospholipids in humans and mice with lesions (Horkko, S. et al (1999) J. Clin.Research 103, 17-128; Palinski, W. et al. (1995) Arterioscler Thromb. Vasc.Biol 15, 1569-1576; Palinski, W. et al. (1996) J. Clin. Invest. 98, 800-814) draw attention to the pathological relevance of these molecules. An increasingly increasing interest has focused on two important representatives in the series of homologous oxidized phospholipids, namely 1-palmtoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1 -palpitoyl-2- (5-oxovaleroyl) -sn-glycerol-3-phosphocholine (POVPC). Its importance is accentuated by the finding that they selectively activate processes in vascular wall cells that may contribute to the pathogenesis of atherosclerosis as well as to other chronic inflammatory diseases. It has been shown that both lipids are enriched 3 to 6 times in rabbit atherosclerotic lesions corresponding to approximately 62 and 16 ng / mg of PGP and POVPC of wet weight in aorta, respectively (Subbanagounder, G. el al. 2000) Arterioscler, Thromb, Vasc.Biol, 20, 2248-2254). In WO 01/75 70 A a method is described for assessing the risk of atherosclerosis, in which a biological sample comprising HDL is contacted with an oxidized phospholipid and the change in the amount of oxidized or non-oxidized phospholipid is measured, wherein the absence of change in the amount of oxidized phospholipid indicates a risk of atherosclerosis. As mentioned above, oxidized phospholipids are involved in the pathogenesis of atherosclerosis. Oxidized phospholipids are also known to be cleaved hydrolytically by enzymes that are associated with lipoproteins and having antiaterogenetic activity, for example phospholipases or PAF acetylhydrolases. It is the object of the present invention to provide compounds that can be used to collect information about the activity of antiaterogenetic enzymes in a sample. In particular, their interaction with enzymes should result in detectable products that allow us to draw conclusions about the enzymatic activity of the sample.

This object is achieved by an oxidized phospholipid having one of the general formulas I or II Where A = O, C, NH, or S; B = O, C, NH, or S; and R2 is selected from the group consisting of -CO- (CH2) n-CH3; -CO- (CH2) n-CHO; and -CO- (CH2) n -COOH, with n = 3-7, with the proviso that in the general formula I, R1 is selected from the group consisting of -CH2- (CH2) n-X; and -CO- (CH2) n -X with n = 5-11, wherein X is a fluorophore; and in general formula II, R1 is selected from a group consisting of -CH = CH- (CH2) n -CH3 with n = 9-15; - (CH2) n-CH3 with n = 11-17; and -CO- (CH2) n -CH3 with n = 10-16; and R3 is selected from the group consisting of -CO- (CH2) n-X; and -SO2- (CH2) n-X, with n = 0-5, wherein X is a fluorophore. According to a referred modality, A and / or B is oxygen. According to another preferred embodiment, in the general formula I, X is a chlorophore selected from the group consisting of pyrene, perylene and nitrobenzaxadiazole and is preferably pyrene. According to another preferred embodiment, in general formula II, X is a fluorophore selected from the group consisting of pyrene, perylene, borodiazaindacene (BODIPY ™), cyanine dye 2, cyanine dye 3, cyanine dye 5 and Alexa dyes. Preferred moieties of oxidized phospholipids according to the general formula I are the following compounds: • 1- (10-pirendecanoyl) -2-glutaroyl-sn-glycero-3-phosphocholine (PyrGPC) 1- (10-pirendecanoyl) -2- (5-oxovaleroyl) -sn-glycero-3-phosphocholine (PyrOVPC) Preferred moieties of oxidized phospholipidphos according to the general formula II are the following compounds: • 1-palmitoyl-2 -glutaroyl-sr »-glycero-3-phospho-A / - (3- [4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen] -propionyl) -ethanolamine (BODIPY-PGPE) • 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho- / V- (Alexa fluoro 647-carbonyl) -ethanolamine (Alexa647-PGPE). • 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-A / - (3- [4,4-d-fluoro-4-bora-3a, 4-diaza-s-ndacen ] -propionyl) -ethanolamine (BODIPY-POPE) · 1-palmitol-2- (5-oxovaleroyl) -sn-glycero-3-phospho-A / - (3- [4,4-difluoro-4-bora] - 3a, 4a-d¡aza-s-nndacen] -prop¡on¡l) -ethanolamine (BODIPY-POVPE) It has been found that fluorescently oxidized phospholipids labeled according to the invention can be used as substrates for enzymes having antiaterogenetic activity. The claimed compounds are particularly useful for the diagnostic determination of these enzymes in blood samples. The fluorescently labeled cleavage products resulting from the interaction of the oxidized phospholipids of the enzymes in question can be easily and sensitively determined by chromatographic analysis such as HPLC and thereby provide information about the enzymatic activities present in the sample. Another aspect of the invention thus relates to the use of the oxidized phospholipid according to the invention for determining the presence of enzymes having antiaterogenetic activity, preferably phospholipases or PAF-acetylhydrolases in a sample. A further aspect of the invention relates to a method for determining the presence of enzymes having antiaterogenetic activity in a sample comprising adding oxidized phospholipid according to the invention to the sample and subjecting the sample to graphical analysis, preferably HPLC. The present invention will be explained in more detail by the following examples and the accompanying drawings.

EXAMPLES Synthesis of fluorescent oxidized phospholipids Preparation of 1-palmitoyl-2-qlutaroyl-sn-glycero-3-phospho-n- (3- [4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen-1-propionyl ) -ethanolamine (BODIPY-PGPE) 1. - Synthesis of 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) SCHEME A PGPC (Scheme A, 1) was synthesized according to a modified version of the Watson et al. (J. Biol. Chem. 272, 13597-13607, 1997) and Subbanagounder et al. (Free Radie, Biol. Med. 28, 1751-1761, 2000). A solution of 1-palmitoyl-sn-glycero-3-phosphocholine (62 mg, 125 μ? T ?? es) dry, glutaric anhydride (70 mg, 613 μ??? 5 eq) and DMAP (p. - (/ \ /, A / '- dimethylamino) pyridine, 75 mg, 614 μg, 5 eq) in 6 ml of anhydrous dichloromethane was stirred magnetically overnight at 35 ° C. The reaction was monitored by TCL with CHCL3 / MeOH / 25% NH3 (65: 35: 5, v / v / v) as a developer system and was quenched by the addition of 3 ml of MeOH. The resulting mixture was washed once with 1.8 mg of MeOH / hbO (1: 1, v / v). After evaporation of the organic phase, the remaining DMAP was removed by trituration with 4 ml of diethyl ether. The removal of the supernatant generated 32 mg of PGPC (42%, Rf = 0.005). 2. Synthesis (te 1 -palmitoyl-2-qlutaroyl-sn-qlycero-3-phosphoethanolamine (PGPE) PGPE (Scheme A, 2) was obtained by the transphosphatidylation of PGPE (90 mg, 148 μ ???? ßß) (Scheme A, 1) by PLD (phospholipase D, 29 units) in one emulsion of 1.4 ml of toluene and 4.3 ml of a buffer solution of 0.5 M sodium acetate (pH 7.2) containing 0.5 M ethanolamine (Scheme A), step 1 ). The diphasic system was stirred at 35 ° C overnight. The reaction was monitored by TLC and quenched by the addition of 4.3 ml of MeOH. The product was extracted with 43 ml of CHCl3 / MeOH (2: 1, v / v). The organic phase was washed twice with 11 ml of MeOH / H 2 O (1: 1, v / v) to remove excess ethanolamine, evaporated and subjected to preparative TLC. The product was scraped and eluted three times from the silica gel with CHCl3 / MeOH (1: 4, v / v). Evaporation of the combined extracts generated the desired product (30.3 mg, 36%, Rf = 0.30). 3. Synthesis of 1-palmitl-2-qlutaroyl-sn-qlycero-3-phospho-N- (3- [4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen-propionyl) -ethanolamine (BODIPY-PGPE) To a magnetically stirred solution of BODIPY-SE (succinimidyl ester of 4,4-difluoro-5,7-d, methyl-4-bora-3a, 4a-diaza-s-nadacene-propionic acid, 0.96 mg, 2.5 μg) in 1 ml of CHCl3 / MeOH (2: 1, v / v) were added PGPE (4.4 mg, 7.8 μ ??β, 3 eq) (Scheme A, 2 ) and triethylamine for (10 μ ?, 72 μ? -noles, 30 eq) (Scheme A, step 2). Then the flask was flushed with nitrogen, protected from the light and the resulting solution was stirred at room temperature for 1 hour. The progress of the reaction was monitored by TLC. The solvent was removed under a stream of nitrogen until the volume of 400 μ ?, was reached, from which the liquid was purified by preparative TLC. The desired compound was visualized under UV light, scraped off the TLC plate and eluted three times from the silica gel with CHCIa / MeOH (1: 4, v / v). The solvent was removed from the combined statuses by rotary evaporation to generate BODIPY-PGPE (1.77 mg, 85%, Rf = 0.28) (Scheme A, 3).

Preparation of 1-palmitoyl-2-qlutaroyl-sn-qlycero-3-phospho-N- (Alexa647-carbonyl) -ethanolamine (Alexa647-PGPE) Alexa647 PGPE corresponds to BODIPY-PGPE (Scheme A, 3) but contains Alexa647 instead of BODIPY as its leading group. Obtained from Alexa647-SE (succinimidyl ester of Alexa Fluor 647 carboxylic acid, 0.5 mg, 0.4 μ? T ??? ßß) and PGPE (0.68 mg, 1.2 μ ???? ßß, 3 eq) (Scheme A, 2 ) in 0.5 ml of CHCl3 / MeOH (2: 1, v / v), after the addition of triethylamine (5 μ ?, 36 μG ???, 90 eq) followed by stirring at room temperature for 90 minutes. The reaction mixture was strictly protected from light. The progress of the reaction was monitored by TLC (RP-18 F254s; Merck, Darrnstadt, Germany). The product was purified by preparative TLC with H20 / EtOH / n-propanol (20:57:23, v / v / v) as a developer system, scraped and eluted twice with CHCl3 / MeOH (1: 1, v. / v) and once with MeOH. Removal of the solvent under a stream of nitrogen provided Alexa647-PGPE (0.26 μGp ??, 65%, Rf = 0.80).

Preparation of 1 -2- (5-oxovaleroyl) -sn-glycero-3-phospho-N- (3-, 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen-1-propionyl) -ethanolamine ( BQDIPY- POVPE) 1. Synthesis of 1-palmitoyl-2- (5-dimethoxypentanoyl) -sn-glycero-3-phosphoethanolamine SCHEME B NoOH OH V OV-o A solution of 5,5-dimethoxy-pentanoic acid methyl ester (220 mg, 1.25 mmol) (Scheme B, 4) and sodium hydroxide (250 mg, 6.25 mmol 5 eq) in 5 ml of H2O / MeOH / THF ( 2; 5: 3, v / v / v) was stirred at room temperature for 90 min (Scheme B, step 1). After cooling to 0 ° C, the reaction mixture was acidified to pH 2.1 by the subsequent addition of 6 ml of 1 N and appropriate amounts of 0.1 N HCl and then extracted with dichloromethane (3 x 15 ml). The combined organic extracts were washed with water (2 x 10 mL) and dried over Na2SO4. The solvent was removed under reduced pressure except for 10-15 ml containing the desired product (Rf = 0.35) (Scheme B, 5), which was used immediately for the acylation reaction without further purification. 1 -palmitoyl-sn-glycero-3-phosphocholine (209 mg, 0.42 mmloes) (Scheme B, 6), DCC (?,? '- dicyclohexylcarbodumide, 270 mg, 1.3 mmol, 3 eq) and DMAP (160 mg, 1.3 mmoles, 3 eq) were added to this solution of 5,5-dimethoxypentanoic acid (Scheme B, step 2). The mixture was stirred under nitrogen at room temperature overnight. The reaction was monitored by TLC. After the addition of 6 ml of MeOH, the organic solution was washed twice with MeOH / H 2 O (1: 1, v / v). The solvent was removed under vacuum, and traces of water were evaporated after the addition of benzene / EtOH (3: 2, v / v). The oily residue was flash chromatographed on 12 g of silica gel with CHCl3 / MeOH / 25% NH3 (65: 35: 5, v / v / v) as a solvent to generate 1-palmitoyl-2- (5-dimethoxypentanoyl) -sn-glycero-3-phosphocholine (245 mg, 91%, Rf = 0.14) (Scheme B, 7). The ethanolamine analogue (Scheme B, 8) was obtained by the transphosphatidylation of choline lipid as previously described for the conversion of PGPC to PGPE (compare Scheme A, step 1) (yield: 6.2 mg, 66%, Rf = 0.24). using CHCl3 / MeOH / 25% NH3, 65: 35: 5, v / v / v as a developed agent ^ (Scheme B, step 3). 2. Synthesis of 1-palmitoyl-2- (5-oxovaleroyl) -sn-qlycero-3-phospho-N- (3- [4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen-1-propionyl) -ethanolamine (BODIPY-POVPE) Triethylamine pa (10 μ ?, 72 μ? t ??? ße, 14 eq) was added to a solution of 1-palmytoyl-2- (5-dimethoxy-pentanoyl) -sn-glycero-3- phosphoethanolamine (3.0 mg, 5.0 μ? t ??? ße) (Scheme B, 8) and BODIPY-SE (1.95 mg, 5.0 μ ????, 1 eq) in 1 ml of CHCl3 / MeOH (2:, v / v) (Scheme B, step 4). The reaction mixture was stirred at room temperature for 80 min. The solvent was removed under vacuum to generate 1-palmitoyl-2-dimethoxy-pentanoyl-sn-glycero-3-phospho-A / -BODIPY-propionyl) -ethanolamine (Scheme B, 9), which was suitable for the next reaction without further purification. The desired product release was achieved by cleaving the acetal from the stable precursor (2.0 mg, 2.3 μ? T ??? ße) with 400 μ? of THF / HCl (1 N) (Scheme B, step 5). After only 2 min, the reaction mixture was neutralized with NaHCO 3 followed by extraction of the product with 1.2 ml of CHC / MeOH (2: 1, v / v). The organic phase was washed twice with 300 μ? of water, dried over Na2SO4 and evaporated under reduced pressure leading to 1.65 mg of BODIPY-POVPE (87%, Rf = 0.22 using CHCl3 / MeOH / H20, 15: 5: 0.1, v / v / v as a developer system ) (Scheme B, 10).

Preparation of 1-palmitol-2-oleoyl-sn-qlycero-3-phospho-N- (3-G4,4-difluoro-4-bora-3a, 4a-diaza.s.indacen] -propionyl) -ethanolamine (BODIPY-POPE) BODIPY-SE (1.00 mg, 2.57 μ? T ??? ße), triethylamine (10 μ ?, 72 μ? -noles, 28 eq) and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 5.53 mg, 7.70 μ ????, 3 eq) were dissolved in 1 ml of CHCl3 / MeOH (2: 1, v / v). The mixture was stirred at room temperature for 60 min, and then the solvent was removed under a stream of nitrogen. The white oily residue was dissolved in 500 μ? of CHCl3 / MeOH (2: 1, v / v). The product was purified by preparative TLC. The fluorescent band containing the product was scraped off the plate and eluted three times with CHCIs / MeOH (1: 4, v / v). The combined extracts were evaporated to generate the desired product (1.31 mg, 51%, Rf = 0.59).

Synthesis of 1- (10-pirendecanoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PyrGPC) SCHEME C ( 1" To a magnetically stirred emulsion of 1,2-bis (10-pirendecanoyl) -sn-glycero-3-phosphocholine (45 mg, 47 μmol) (Scheme C, 11) in a mixture of 3 ml of 0.1 M buffer solution. -HCl (pH 8) containing 0.1 M CaCl2 and 3 ml of diethyl ether (free of peroxide) were added 50 units of PLA2 (poison Naja naja) (Scheme C, step 1). The reaction mixture was stirred overnight at 35-40 ° C. After removing the diethyl ether, the product was extracted from the aqueous solution with CHCl3 / MeOH (2: 1, v / v) (3 x 5 mL). The combined organic fractions were evaporated and the residual water was removed under high vacuum leading to 50 mg of a mixture of 1- (10-pirendecanoyl) -sn-glycero-3-phosphocholine (Scheme C, 12) and free pyrendecanoic acid. The latter was removed by trituration with diethyl ether. After removal of the solvent under vacuum, 25 mg of the pure lysophospholipid were obtained (88%, Rf = 0.12 in CHCl 3 MeOH / AcOH / H 2 O, 50: 30: 10: 5, v / v / v / v as a developer system ). Glutaric anhydride (12 mg, 105 μ? T ???, 11 eq) and anhydrous DMAP (4 mg, 33 μ? ßßß, 3 eq) were added to the lysophospholipid solution (6.0 mg, 9.8 μg) ? t ???) in 3 ml of anhydrous dichloromethane (Scheme C, step 2a). The reaction was stirred overnight at 35-40 ° C. Flash chromatography of the crude product on 10 g of silica gel with CHCl3 / MeOH / H2O (65: 25: 4), v / v / v) led to PyrGPC (2.2 mg, 31%, Rf = 0.18 in CHCl3 / MeOH / H2O, 50: 30; 10: 5, v / v / v / v as a developer system) (Scheme C, 14).

Synthesis of 1- (10-pirendecanoyl) -2-85-oxovaleroyl) -sn-qlycero-3-phosphocholine / PyrOVPC) 1- (10-pirendecanoyl) -sn-glycero-3-phosphocholine (10 mg, 16 μ? ?? ße) (Scheme C, 12) was acylated with 5,5-dimethoxy-pentanoic acid ("5" acid) in 10 ml of dichloromethane containing DCC (200 mg, 1.0 mmol, 59 eq) and DMAP (200 mg, 1.6 mmol, 100 eq). The reaction mixture was stirred at room temperature overnight (Scheme C, step 2b). The progress of the reaction was monitored by TLC until the reaction was stopped by the addition of 6 ml of MeOH. The resulting solution was washed twice with MeOH / H 2 O (1: 1, v / v), and the solvent was removed under reduced pressure. The crude product was purified by preparative TLC, scraped and eluted 5 times with 4 ml of CHCl3 / MeOH (1: 4, v / v). The residual silica gel was removed by washing the combined organic fractions (concentrated to a volume of 5 ml) with 1 ml of MeOH / H 2 O (1: 1, v / v). Evaporation of the solvent generated 6 mg of 1- (10-pirendecanoyl) -2- (5-dimethoxy-pentanoyl) -sn-glycero-3-phosphocholine (40%, Rf = 0.1 7) (Scheme C, 15). Deprotection of intermediate 15 (5.6 mg, 7.4 μ ????) was achieved by cleavage of acetal with THF (HCl (1 N), followed by neutralization with NaHCO3 and extraction of product with CHCl3MeOH (2: 1, v / v) The organic phase was washed twice, dried over Na2SO4 and evaporated under reduced pressure to yield 1.1 mg of PyrOVPC (21%, Rf = 0.05 (Scheme C, step 3).

Mass Spectrometry Mass spectra were performed with laser defection assisted by diagonal matrix ionization time-of-flight (MALDI-TOF) in a Micromass TofSpec2E equipped with a nitrogen laser (? = 337 nm, operated at 5 Hz) and a unit of approach with time delay. The ions were generated by irradiation just above the threshold laser power. Spectra were recorded in the reflectron mode with an acceleration voltage of 20 kV and externally calibrated with a suitable mixture of polyethylene glycols (PEG). Samples were typically prepared by mixing solutions of the matrix (2,5-dihydroxybenzoic acid, c = 10 mg / ml, CH3CN / 0.1% trifluoroacetic acid (TFA), 70:30, v / v, the analyte (c = 0.01 -1 mg / ml, CHCl3MeOH, 2: 1, v / v) and NaTFA (c = 1 mg / ml, CH3CN / H20, 70:30, v / v) in a ratio of 10: 1: 0.5 (v / v / v) A 0.5 μl aliquot of the mixture was deposited on the sample plate (stainless steel) and allowed to dry under air.The spectra of 50-100 shots were averaged to improve the signal-to-noise ratio. the m / z values discussed in this work correspond to the most intense peak of any isotope distribution.The mass spectra of BODIPY-POVPE, BODIPY-PGPE and BODIPY-POPE are shown in Figures 1A-1C. PyrGPC and PyrOVPC are shown in Figures 2A-2B.

Claims (15)

NOVELTY OF THE INVENTION CLAIMS

1. - An oxidized phospholipid having one of the general formulas I or II where A = O, C, NH or S; B = O, C, NH or S; and R2 is selected from the group consisting of -CO- (CH2) n-CH3; -CO- (CH2) n-CHO; and -CO- (CH2) n -COOH, with n = 3-7, with the proviso that in general formula I, Ri is selected from the group consisting of -CH2- (CH2) n-X; and -CO- (CH2) n-X; with n = 5-11, wherein X is a fluorophore; and in the general formula II R-i is selected from the group consisting of -CH = CH- (CH2) n -CH3 with n = 9-15; - (CH2) n-CH3 with n = 11-17; and - CO- (CH2) n-CH3 with n = 10-16; and R3 is selected from the group consisting of -CO- (CH2) n -X; and -SO2- (CH2) n-X, with n = 0-5, wherein X is a fluorophore.

2. The oxidized phospholipid of general formula I according to claim 1, further characterized in that A and / or B is oxygen.

3. The oxidized phospholipid of general formula I according to claim 1 or 2, further characterized in that X is a fluorophore selected from the group consisting of pyrene, perylene and nitrobenzaxadiaxol, and is preferably pyrene.

4. The oxidized phospholipid of general formula I according to any of claims 1 to 3, further characterized in that it is 1- (10 pirendecanoiH) -2-glutaroyl-sn-glycero-3-phosphocholine (PyrGPC).

5. - The oxidized phospholipid of general formula I according to any of claims 1 to 3, further characterized in that it is 1- (10-pirendecanoyl1) -2- (5-oxovaleryl) l-sn-glycero-3-phosphocholine ( PyrOVPC).

6. The use of the oxidized phospholipid of general formula I according to any of claims 1 to 5 to determine the presence of enzymes having antiaterogenetic activity, preferably phospholipases or PAF-acetylhydrolases, in a sample.

7. A method for determining the presence of enzymes having antiaterogenetic activity in a sample comprising adding oxidized phospholipid of general formula I according to any of claims 1 to 5 to the sample and subjecting the sample to chromatographic analysis, preferably HPLC .

8. - The oxidized phospholipid of general formula II according to claim 1, further characterized in that A and / or B is oxygen.

9. - The oxidized phospholipid of general formula II according to claim 1 or 8, further characterized in that X is a fluorophore selected from the group consisting of pyrene, perylene, borodiazaindacene (BODIPY ™), cyanine dye 2, cyanine dye 3, cyanine dye 5 and Alexa dyes.

10. - The oxidized phospholipid of general formula II according to any of claims 1, 8 or 9, further characterized in that it is 1 -palmitoyl-2-glutaroyl-sn-glycero-3-phospho- / V- (3- [ 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen] -propionyl) -ethanolamine (BODIPY-PGPE).

11. - The oxidized phospholipid of general formula II according to any of claims 1, 8 or 9, further characterized because it is 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-A / - (Alexa Fluor 647 -carbonyl) -ethanolamine (Alexa647-PGPE).

12. - The oxidized phospholipid of general formula II according to any of claims 1, 8 or 9, further characterized in that it is 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-A / - (3- [ 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen] -propionyl) -ethanolamine (BODIPY-POPE).

13. The oxidized phospholipid of general formula II according to any of claims 1, 8 or 9, further characterized in that it is 1-palmitoyl-2- (5-oxovaleroyl) -sn-glycero-3-phospho-A / - (3- [4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen] -propionyl) -ethanolamine (BODIPY-POVPE).

14. The use of oxidized phospholipid of general formula II according to any of claims 1, 8 to 13 to determine the presence of enzymes having antiaterogenetic activity, preferably phospholipases or PAF-acetylhydrolases, in a sample.

15. - A method for determining the presence of enzymes having antiaterogenetic activity in a sample comprising adding oxidized phospholipid of general formula II according to any of claims 1, 8 to 13 to the sample and subjecting the sample to chromatographic analysis, preferably HPLC.