coronary heart disease ( chd ) is the major source of mortality in the western world . although it was believed that this is principally a problem of the developed world , new 
 evidence , provided by the world health organization , shows that cardiovascular 
 disease will become a major problem in developing countries as well , unless 
 they stop acquiring western nutritional habits . heart attack and stroke are the 
 clinical results of a systemic vascular process widely known as atherosclerosis 
 . atherosclerosis can be described as a systemic vascular process consisting 
 microscopically of foam cell formation in the subendothelial space , inevitably 
 leading to fatty streak and then fibrous plaque formation . the importance of mediterranean diet on preventing chd has been 
 indicated   by several studies [ 35 ] . olive oil is the main 
 source of fat in the aforementioned diet , thus its beneficiary 
 effects on health have been widely examined , both in vitro and in 
 vivo [ 47 ] . olive oil is 
 extracted from olive fruits by pressure , thus resulting into two wastes : olive 
 mill wastewater ( omw ) and olive pomace ( p ) [ 8 , 9 ] . olive pomace or just pomace 
 still retains a small amount of olive oil and mainly consists of water , olive 
 skin , olive flesh , and pit fragments ; therefore , its major ingredients are 
 sugars ( mainly polysaccharides ) , proteins , fatty acids ( oleic acid and other 
 c2c7 fatty acids ) , polyalcohols , polyphenols , and other pigments [ 912 ] . it is mainly used for extracting olive pomace oil ( secondary value oil ) [ 12 , 13 ] ; however , it can be used as a fuel as well . according to our theory , platelet activating factor 
 ( paf ) is implicated in atherogenesis   and this is also supported by other 
 researchers [ 1518 ] . the increase of paf levels and activity in blood , which 
 occurs both in pathological inflammatory conditions and during oxidative stress 
 through ldl and membrane phospholipid oxidation , is believed to be of crucial 
 importance for the initiation of atherosclerosis . paf activity can be 
 controlled either by paf - ah or by paf antagonists   derived either from food or produced endogenous . such 
 paf antagonists have been isolated from various food stuffs like yogurt , honey , wine [ 2123 ] , fish [ 24 , 25 ] , and olive oil [ 4 , 26 ] , which 
 constitute food stuffs of the mediterranean diet , thus enhancing our 
 understanding of this diet 's beneficial effects on health . our group has 
 previously reported the inhibitory effect of olive oil on experimental 
 atherosclerosis induction in rabbits . it was , also , demonstrated that this 
 inhibitory effect is mainly attributed to oopl , mainly containing paf 
 antagonists , rather than to olive oil neutral lipids ( oonls ) , mainly containing 
 oleic acid and antioxidants . since , on one 
 hand , a percentage of olive oil is still retained in olive pomace   and 
 on the other hand olive pomace , as a waste of olive oil industry , has low 
 interest value , the existence of paf antagonists in olive pomace , could 
 contribute to increase its biological value . the existence of paf antagonists 
 in olive pomace polar lipid ( ppl ) extract has already been reported and the 
 bioactive compound has been chemically characterized as a 
 glycerylether - sn-2-acetyl glycolipid sharing structural features with the 
 respective one of oopl . the aim of this study is to examine in vivo the 
 antithrombotic and antiatherosclerotic activity of ppl on cholesterol fed rabbits and 
 to compare it with the effect of oopl . solvents of analytical grade and for high - performance liquid 
 chromatography ( hplc ) and gas chromatography ( gc ) analyses , as 
 well as silicic acid , 3570 meshes , astm 7733 for column 
 chromatography , were supplied from merck ( darmstadt , germany ) . 
 bovine serum albumin ( bsa ) , paf 
 ( 1-o - hexadecyl-2-acetyl - sn - glycero-3-phosphocholine ) , lipid 
 standards for hplc and gc , trichloroacetic acid ( tca ) , cholesterol 
 ( 95% ( gc ) , equivalent to usp / nf ) , naphthalene 
 ( scintillation grade ) , and histopaque 1077 were obtained from 
 sigma ( st . louis , mont , usa ) . [ h]-acetyl paf ( 10 ci / mmol ) 
 ( net 910 ) was purchased from nen ( dupont , boston , mass , usa ) . 2,5-diphenyloxazole ( ppo ) and 1,4-bis(5-phenyl-2-oxazolyl ) benzene 
 ( popop ) were purchased from bdh chemicals ( dorset , england ) . scintillation liquid cocktail ( dioxane base ) was prepared by 
 diluting 7 g ppo , 0.3 g popop , and 100 g napthalene in 200 ml 
 h2o and then transferred to 1 l of dioxane . virgin olive oil 
 of koroneiki variety extracted by pressure and its respective 
 olive pomace came from kalamata , greece . olive oil and pomace were subjected to a modified counter current distribution . according 
 to this method , the ethanol phase at the end yields 96% of the polar lipids as 
 evaluated with [ h]-paf , whereas the combined petroleum ether 
 phases contain the neutral lipid class . this modified counter current distribution 
 is normally applied in vegetable oils [ 4 , 29 ] . briefly , every 100 ml of olive 
 oil were placed in a separatory funnel and diluted in 400 ml of petroleum ether 
 ( pe ) solvent , thus constituting the initial total volume of 500 ml . after 5 minutes , the 
 two phases were separated and a new amount of 100 ml ethanol solvent was added 
 in the equilibrated upper phase of pe extract , followed by the equilibration 
 and separation of the two phases . the whole procedure was repeated three times 
 totally , thus obtaining at the end the oopl in ethanol extracts of final volume 
 300 ml . a modified extraction ( figure 1 ) of the aforementioned extraction procedure was applied on 
 olive pomace . briefly , for every 100 g of olive pomace , 250 ml of pe solvent were 
 added . after 5 minutes in stirring , the liquid is separated from olive pomace 
 through buchner filtering . the procedure was repeated once more in the 
 remaining olive pomace thus giving 500 ml of pe extract in total , which were 
 placed in a funnel containing 250 ml of ethanol solvent . the two phases were 
 equilibrated and then separated . in the remaining residue of olive pomace , 
 250 ml of ethanol solvent were added twice and after 5 minutes in stirring , both 
 liquids were removed through buchner filtering . ethanol extracts ( 500 ml ) were 
 then placed in a funnel containing 250 ml of pe solvent and the two phases were 
 separated after equilibration . all ethanol phases , of 750 ml total volume , were 
 pooled together , thus consisting the polar lipid extract of olive pomace ( ppl ) , 
 while all pe phases , also 750 ml total volume , consisted the neutral lipid 
 extract , which was not further used . the evaluation 
 of the above method concerning the recovery of polar lipids from olive pomace 
 was performed with [ h]-paf . 4070c ) and ethanol 87% solvents were preequilibrated 
 with each other , prior use in extraction procedures . washed rabbit 
 platelets were prepared as previously described 
 . the examined samples were 
 dissolved in absolute ethanol and bsa 2.5 mg / ml saline was added to give a 
 ratio of ethanol / bsa 1/19 . the platelet aggregation induced by paf ( 2.5  10 m , final concentration ) was measured as paf - induced aggregation , in washed 
 rabbit platelets , before ( considered as 0% inhibition ) and after the addition 
 of various concentrations of the examined sample as previously described . 
 consequently , the plot of percent inhibition ( ranging from 0 to 100% ) versus 
 different concentrations of the sample is linear . from this curve , the 
 concentration of the sample that inhibited 50% paf - induced aggregation was 
 calculated , and this value was defined as ic50 . buffer control data 
 were performed with 2.5 mg of bsa per 1 ml saline and the results were appropriately 
 corrected . in addition , various concentrations of each examined sample were 
 added into the aggregometer cuvette . according to cross - desensitization 
 experiments that were performed , platelets were desensitized by the addition of 
 the examined lipid at a concentration that caused reversible aggregation . 
 second stimulation with paf the study was performed using a chronolog aggregometer ( model 
 400 ) ( havertown , pa , usa ) coupled to a chronolog recorder at 37c with constant stirring at 
 1200 rpm . the experiments involving the specific binding of [ h]-paf to washed rabbit platelets and its inhibition by oopl and ppl were performed as previously described with some modifications . briefly , [ h]-paf ( 1 nm ) , paf ( 1 m ) , oopl , and ppl were diluted in 
 absolute ethanol , and bsa 10 mg / ml in tg - ca 
 buffer ph 7.2 was added to give a ratio of ethanol / bsa 1/19 . incubations were carried out in a total 
 0.5 ml volume . in 360 l , tg buffer ph 7.2 contained cacl2 1.72 mm , [ h]-paf ( 1 nm ) in 
 the presence or absence of either paf ( 1 m ) , oopl , or ppl were added and the volume was adjusted in 460 l with bsa 10 mg / ml in tg - ca buffer ph 7.2 . then , 40 l of 
 washed rabbit platelets ( 1  10 platelets ) in tg buffer ph 6.2 were added to start the incubation time . double 
 incubations were carried out at 22c for 4 minutes , a time period where [ h]-paf 
 degradation was calculated to be less than 1% . termination was performed with 
 the addition of 0.7 ml ice - cold tg - ca buffer ph 7.2 , followed by rapid centrifugation at 11500  g for 2 minutes at 4c . the specific paf 
 antagonist , bn 52021 , was used as a reference compound . the radioactivity was 
 measured by scintillation counting on a 1209 rackbeta liquid scintillation 
 counter from pharmacia ( wallac , finland ) . nonspecific binding was defined as the total binding measured in the presence 
 of excess unlabelled paf ( 1 m ) and specific binding was defined as the difference between total binding and nonspecific binding . the percent inhibition was expressed as % i = ( total binding  total binding with tested compound)/specific binding  100 . the 
 ic50 value was defined as the concentration of inhibitor required 
 obtaining 50% inhibition of the paf specific binding . scatchard analysis was 
 performed with graphpad prism software ( san diego , calif , usa ) . briefly , samples were dried under a stream of nitrogen and dissolved in 3.5 ml of water . an amount of 0.1 ml folin - ciocalteu reagent was added , followed 
 after 3 minutes by addition of 0.4 ml of 35% aqueous na2co3 . the reaction mixture was kept for 1 hour , and the intensity of the blue color was measured at 725 nm . gc analysis was carried out on a shimadzu model gc-17a gas 
 chromatograph ( kyoto , japan ) , using 
 an agilent j & w db-23 capillary column 
 ( 60 m  0.25 mm , i.e. , 0.25 m ) purchased by agilent 
 technologies ( santa clara , calif , usa ) . both samples were dried under a stream of nitrogen and then diluted in 1 ml of hexane . methyl - esterification was performed by the addition of 4 ml of koh 0.5 n in 
 methanol / water ( 90/10 ) at room temperature for 5 minutes under continuous 
 vortexing . after that the solution was neutralized by the addition of hcl 6n 
 and 2 ml of h2o were added and the fatty acid methylesters were 
 extracted twice by 2 ml hexane each time . oven thermal program started at 120c and remained at this 
 temperature for 5 minutes , then 
 rose at 180c within 6 minutes , followed by a temperature increase at 220c within 2 minutes . eighteen healthy male new zealand rabbits of specific weight and age were purchased from a commercial breeder and were individually housed in atomic stainless steel cages in constant conditions of temperature ( 19 + 1c ) , relative moisture ( 55 + 5% ) , and air conditioning ( 12 full changes of air per 1 hour ) . rabbits were randomly divided into three groups of six animals each and were given specific diet for 22 days . group a was given atherogenic diet , while groups b and c were 
 given atherogenic diet enriched with oopl ( 0.16% w / w ) and ppl 
 ( 0.13% w / w ) , respectively . their food was prepared freshly every three days before 
 consumption . on the 22nd day , rabbits were given xylazine ( rompun , bayer , leverkusen , 
 germany ) 5 mg / kg body weight and cetamine ( fort dodge laboratories inc . , fort dodge , iowa , usa ) 25 mg / kg body weight 
 intramuscularly . rabbits fell unconscious and soon afterwards euthanasia took 
 place by injecting pentothal ( hospital products division , abbott laboratories abbott park , ill , usa ) 20 mg / kg body weight , intravenously . through a median longitudinal incision , the thoracic and peritoneal cavities were opened and the aorta was dissected from the aortic valve down to the aortic bifurcation . at the beginning ( 0 days ) and at the end of the experimental time ( 22 days ) , blood was collected 
 from all rabbits through the main ear artery and was placed in polyethylene 
 tubes containing anticoagulant with a ratio of blood / anticoagulant 9 : 1 ( v / v ) . platelet - rich plasma ( prp ) was obtained by centrifugation of blood samples at 562    g for 13 minutes , while platelet - poor plasma ( ppp ) was obtained by further centrifuging the specimens at 1750  g for 20 minutes . the centrifugation was performed on heraeus labofuge 400r ( hanau , germany ) at 24c . aliquots of paf solution in chloroform / methanol ( 1 : 1 v / v ) were evaporated under a stream of 
 nitrogen , and were redissolved in bsa ( 0.25% in saline ) in order to obtain paf 
 solutions with final concentrations ranging from   1.0    10 
  to 1.0    10 m. the maximum reversible or the minimum irreversible 
 paf - induced platelet aggregation was determined as the 100% aggregation , and 
 then various paf concentrations were added , so as to achieve aggregations 
 between 20% and 80% . these paf - induced aggregations were of linear response to the respective paf concentration ; therefore , the ec50 value was 
 calculated . these studies were performed using a chronolog aggregometer 
 ( model 400 ) coupled to a chronolog recorder at 37c with constant stirring at 1200 rpm . an amount of paf was 
 dissolved in chloroform / methanol ( 1 : 1 ; v / v ) and mixed with an appropriate 
 amount of [ h]-acetyl - paf . the mixture was dried under a stream of 
 nitrogen and redissolved in bsa ( 10 g/l in tris - hcl buffer ph 7.4 ) , giving the [ h]-acetyl paf solution with a final concentration of 800 m and a specific activity of 2500 cpm / nmol that was used as the substrate of 
 paf - ah . ppp was then placed in tris - hcl 50 mm ph 7.4 with a ratio of 3/2000 at 
 200 l final volume . the whole mixture was incubated at 37c for 2 minutes and the reaction was initiated by adding 5 l of 800 m [ h]-acetyl 
 paf / paf solution in bsa ( 10 g/l in tris - hcl buffer ph 7.4 ) . the reaction took 
 place at 37c for 15 minutes and stopped by binding the unreacted [ h]-acetyl 
 paf with an excess of bsa solution ( 0.75 mg / ml , final concentration ) following 
 by precipitation with tca with a final concentration of 9.6% ( v / v ) . the mixture 
 was placed in ice bath for 15 minutes and then was centrifuged at 16,000  g for 
 5 minutes , at 4c . the [ h]-acetate released from the reaction was 
 measured on a liquid scintillation counter ( 1209 rackbeta , pharmacia ) by 
 placing 0.1 ml of the suspension liquid into 5.0 ml of scintillation liquid 
 cocktail ( dioxane base ) . the 
 enzyme activity was expressed as nmol of paf degraded per minute per l plasma . determination of free and bound paf levels was carried out according to demopoulos et al . . briefly , 5 ml of blood was collected into glass centrifuge tubes containing 20 ml of cold absolute ethanol . the whole is centrifuged at 280  g for 20 minutes , 
 at 4c . the supernatant liquid ( containing free paf levels ) was then separated 
 from the cellular pellet ( bound paf levels ) and both samples were subjected to 
 extraction according to the bligh - dyer method . the total lipid fraction 
 from both samples was then further separated into lipid classes using glass 
 column liquid chromatography ( 20 cm  1 mm i.d . ) . the lipid class containing the 
 polar lipid class ( including paf ) was then purified through hplc using a cation 
 exchange column , scx partisil 10 m 25  4.6 cm i.d . purchased by whatman ( maidstone , uk ) 
 at room temperature , where paf was collected at approximately 30 minutes . 
 the exact paf concentration in the specimen was then measured through 
 biological assay on washed rabbit platelets , where the induced aggregation 
 versus known synthetic paf concentrations is linear . the analysis was 
 performed on a hewlett - packard series 1100 ( avondale , pa , usa ) , equipped with a 100 l loop 
 rheodyne ( i 7725 ) injector . the spectrometer was connected to a hewlett - packard ( avondale , pa , usa ) model hp-3396a integrator 
 plotter . all necessary centrifugations were performed on heraeus labofuge 400r 
 ( hanau , germany ) . plasma total cholesterol ( tc ) , ldl cholesterol ( ldl - c ) , hdl cholesterol ( hdl - c ) , and 
 triglyceride ( tg ) concentrations were measured by standard commercial enzymatic 
 methods ( biomerieux , lyon , france ) using a parallel - multichannel analyzer 
 ( type 7170a , hitachi , tokyo , japan ) . thoracic aorta specimens were fixed in 10% buffered formaldehyde solution , sectioned and 
 embedded in paraffin wax using conventional techniques . for the histopathological examination 5 m thickness tissue slide sections were then cut , transferred on slides and stained with haematoxylin and eosin ( h - e ) . conventional measurements of early atherosclerosis lesions in the histopathological tissue 
 sections of resected aortas were performed , using an automated image analysis 
 system . the apparatus comprised a sony - exwave had color video camera ( sony 
 corporation , japan ) , fitted to a zeiss axiostar light microscope ( zeiss , 
 germany ) , a host computer ( pentium 90 mhz , 32 mb ram ) and a sigmascan version 
 2.0 image analysis software ( jandel scientific , erkrath , germany ) . foam cell 
 formation is characteristic of the early atherosclerosis lesions . in the 
 present study , early atherosclerosis lesions results from ex vivo 
 experiments were expressed as median ( 25th percentile-75th percentile ) . the 
 mann - whitney u - test was performed to assess differences among different groups . the wilcoxon sign test was used to assess differences in the same group at 
 different time intervals . results from in vivo experiments concerning food 
 consumption , rabbit weight , and body weight increase in all three groups as 
 well as ic50 values and were expressed as mean  standard deviation 
 ( sd ) . the t - test was performed to assess differences between ic50 values , food consumption , rabbit weight , body weight increase , and thickness 
 and surface of early atherosclerosis lesions among all three groups . data were analyzed using a statistical software package ( spss for windows , 10.0.1 , 1999 , spss inc . , chicago , ill , usa ) . modified counter current distribution was used as extraction method in order to obtain the two 
 lipid classes oopl and ppl , from olive oil and pomace , respectively . this 
 method has been successfully used on vegetable oils . in order to evaluate 
 the method 's suitability concerning the recovery of ppl , pooled ethanol phases , of 750 ml total volume , yield 97% of polar 
 lipids as evaluated with [ h]-paf . olive oil polar lipids ( oopls ) and pomace polar lipids ( ppls ) 
 derived , using the modified counter current distribution , from 
 olive oil and pomace , respectively , were weighted and subjected to 
 chemical determinations . chemical determinations for total sugars , 
 esters , and phenolic compounds were positive , while phosphorus was 
 not detected ( table 1 ) . these results are in accordance 
 with the ones already reported in our previous work . gc 
 fatty acid analysis performed on oopl showed the existence of 
 palmitic ( 16 : 0 ) , palmitoleic ( 16 : 1 ) , stearic ( 18 : 0 ) , oleic 
 ( cis 18 : 1 ) , linoleic ( 18 : 2 ) , and -linolenic ( 18 : 3 ) fatty acids , whereas in ppl palmitic(16 : 0 ) , oleic ( cis 18 : 1 ) , and -linolenic ( 18 : 3 ) fatty acids were detected . the results expressed as ( % ) of the total oopl and ppl are 
 presented in table 1 . the above extracts were tested in 
 vitro for inhibition of paf - induced platelet aggregation . the aforementioned 
 inhibitory activity was retained for a wide range of tested 
 amounts ranging from 0.01 ng to 1.12 g . the most diluted 
 concentrations of oopl and ppl that resulted in 50% 
 inhibition of paf activity were 1.5(0.009 )  
 10 m and 1.1(0.004 )  10 m , 
 respectively , based on sugar determination . final concentrations 
 are given based on sugar determination since the bioactive 
 microconstituents from both extracts have been characterized as 
 glycolipids [ 4 , 28 ] . statistical analysis showed that oopl 
 ic50 value was higher ( p < .05 ) than ppl ic50 value . consequently , [ h ] paf binding assay on washed rabbit 
 platelets was studied in both absence and presence of unlabelled 
 paf as well as in the presence of oopl , ppl , and bn52021 as a 
 reference compound . scatchard analysis of binding data indicates the presence of two 
 populations of binding sites : one with high affinity ( specific 
 binding ) with an equilibrium dissociation constant kd = 
  2.75  1.16 nm and a maximum number of binding sites bmax = 
  391  112 fmol/10 platelets ( 2355  674 sites per 
 platelet ) and another one with low affinity ( nonspecific binding ) 
 with an equilibrium dissociation constant kd = 8.01  2.47 nm 
 and a maximum number of binding sites bmax = 
  739  122 fmol/10 platelets ( 4450  735 sites per 
 platelet ) . our results show that oopl and ppl inhibit specific paf binding on 
 rabbit platelets . the specific paf receptor antagonist , bn 52021 , 
 inhibited paf binding on rabbit platelets at a concentration of 
 2.3(0.8 )  10 m. in the same way , the most 
 diluted concentrations of oopl and ppl that resulted in 50% 
 inhibition of paf binding were   1.5(0.2 )  10 m 
 and 0.42(0.11 )  10 m , respectively , based on 
 sugar determination . rabbit weight at the beginning ( 0 days ) and at the end ( 22 days ) 
 of the experiment , as well as the daily food consumption , is 
 presented in table 2 . body weight in each group was 
 significantly increased at the end of the experimental time 
  ( p < .05 ) . no statistical difference was detected in rabbit weight 
 among groups in both time intervals . in addition , all groups 
 consumed equal amounts of food ( p > .05 ) . at the end of the experimental time plasma tc , hdl - c , ldl - c levels 
 were significantly increased in all rabbits , while tg levels in 
 plasma were significantly increased only in group a 
 ( table 3 ) . at the end of the experimental time , ec50 values were 
 significantly increased only in groups b and c , while in group a 
 these values were significantly decreased , compared to those of 
 the initial time . significant differences were also observed in ec50 values of 
 groups b and c compared to those of group a ( table 4 ) . paf levels , both free and bound , measured in rabbit blood and 
 plasma paf - ah activity were significantly elevated   ( p < .05 ) at 
 the end of the experimental time ( 22 days ) in all groups ( table 4 ) . the present study was focused on the early steps of atherogenesis , characterized by foam cell formation , where paf , as the most active inflammatory lipid mediator , is believed to play critical role . the early atherosclerosis lesions , which were observed as foam cell layers and assessed as thickness and surface area developed inside the blood vessels , are presented in table 5 . representative pictures of the morphometric analysis performed on the aorta specimens appear in figure 2 , where atherosclerosis lesions ( foam cell layers ) are indicated with arrows . statistical analysis on the morphometric assessment data concerning thickness and surface of early atherosclerosis lesions in groups revealed that rabbits consumed atherogenic diet enriched in oopl or ppl ( groups b and c , resp . ) developed lower early atherosclerosis lesions than rabbits consumed atherogenic diet only ( group a )   ( p < .05 ) . moreover , the atherosclerosis lesions in group c were also lower than those noted in group b ( p < .05 ) . olive oil , over the years , has been the subject of many in vivo studies , concerning its antiatherosclerotic effect . de la cruz et al . reported that virgin olive oil administration to cholesterol - fed rabbits reduced vascular thrombogenicity of the subendothelium , increased antithrombotic activity in the endothelium , and decreased foam cell nuclear count and percentage of the wall occupied by foam cells . . showed that olive oil administration on atherosclerotic rabbits , although does not regress the phenomenon , is able to stop further the progression of atherosclerosis process . the beneficial effects of olive oil were initially attributed to oleic acid and mufas , in general , through their action as antioxidants . recent studies reported that olive oil anti - atherosclerotic effect might also be attributed to microconstituents of olive oil , such as phenolic compounds or phenolic glycosides [ 4 , 5 , 42 ] . paf has been implicated in atherosclerosis [ 2 , 1518 ] while specific paf - antagonists ( bn 52021 ) as well as the ones extracted from olive oil inhibited atherosclerosis development on cholesterol fed rabbits [ 27 , 43 ] . in the present study , the extraction process yielded 97% 
 of pomace polar lipids as evaluated with [ h]-paf that shows 
 the method 's suitability for solid samples . total sugar 
 determination showed that both extracts contain equal amount of 
 sugars , expressed as glucose , and for this reason ic50 values 
 expression was based on this determination . phenolic compound 
 determination showed that ppl contains almost twice the phenolic 
 compound amount than oopl . this was of no great surprise , since 
 pomace has a higher concentration in olive skin and pigments , both 
 rich in phenolic compounds [ 911 ] , than olive oil . concerning fatty acid composition , pomace oil has been reported to 
 have pretty much the same fatty acid composition as olive oil . this was expected , since pomace retains olive oil , which is 
 further extracted using hexane , thus producing pomace oil mainly 
 containing pomace fatty acids . in the present study , 
 fatty acid concentrations in ppl were significantly lower than the 
 respective ones in oopl . in addition , palmitoleic , stearic , and 
 linoleic fatty acids were only detected in oopl . palmitic acid can 
 be found in both oopl and ppl , in approximately the same 
 concentration . on the other hand , oleic and -linolenic acid concentrations in ppl are lower than the ones in oopl . in a recent work , olive pomace lipid extract has been purified on hplc and the most potent paf antagonist derived from this procedure was characterized as glycerylether - sn-2-acetyl glycolipid based on chemical determinations and esms analysis . this active compound shares similar biological activity , chromatographic behavior , and possibly structure to the one from olive oil polar lipids . ppl inhibition potency is higher than that of oopl as indicated by their ic50 values , which may be attributed to their higher phenolic compound concentration whose antithrombotic activity has already been reported [ 21 , 45 ] . paf binding assays performed on rabbit platelets showed that both polar lipid extracts are specific paf receptor antagonists . in a similar way , the aforementioned results indicate that these extracts exert their inhibitory effect on paf actions by blocking paf receptor . enrichment of group b atherogenic diet with oopl was based on previous studies [ 7 , 27 ] . the amount of ppl , which enriched group c atherogenic diet , was equivalent to that of oopl according to their ic50 values . conflicting results , concerning lipid profile of rabbits fed with atherogenic diet enriched in olive oil , exist in previous studies [ 7 , 27 ] . all these data indicate that lipid profile may supply valuable information on the total condition of experimental models ; however , it can not provide information on the atherosclerosis progression and blood vessel condition . platelet aggregation ability as indicated by the ec50 values decreased in groups b and c while increased in group a. no significant difference was observed in the ec50 values between groups b and c after food consumption . these findings suggest that paf - antagonists present in oopl and ppl may be bioavailable in rabbit blood where they can exhibit anti - paf activity . our findings are in accordance with those of previous studies , in which ec50 values increased only in rabbit groups that had consumed atherogenic diet enriched either in olive oil or olive oil polar lipids [ 7 , 27 ] . paf levels have been shown to be elevated in various inflammatory diseases , such as allergy , asthma , sepsis , trauma , shock , acute pancreatitis , and diabetes . in turn , paf molecule can also promote oxidation , thus decisively contributing to atherosclerosis initiation and progression , as has been proposed by our group . in this study , levels of paf , both the one loosely bound to blood components ( defined as  free  ) as well as the one strongly bound to blood components and cell - associated ( defined as  bound  ) , were significantly elevated in all three rabbit groups . it is well known that a variety of cells such as platelets , monocytes , macrophages , and so on biosynthesize paf and paf - like lipids under atherogenic conditions , as well as the produced paf and paf - like lipids amplify cell activation , which in turn further increases paf levels [ 2 , 15 , 46 , 47 ] . paf - ah is the key enzyme to paf degradation and its activity significantly increases during this study in all three groups . the observed elevation may be attributed to the increased paf levels since inflammatory mediators in vivo induce paf - ah activity and paf stimulates enzyme expression . paf - ah is suggested to serve either as a protective enzyme against acute inflammatory and oxidative factors , such as paf , or as a marker , concerning both disease progression and severity , in long term oxidative stress conditions [ 49 , 50 ] . in addition , overexpression of paf - ah appears to induce anti - atherogenic properties in animal model . rabbits belonging to groups b and c developed early atherosclerosis lesions , at significantly lower degree , compared to group a. this is in accordance with our previous study in which the antiatherosclerotic effect of olive oil on cholesterol fed rabbits was attributed to its polar lipid extract ( oopl ) . significant differences were also noted in the early atherosclerosis lesions of group c compared to those of group b , indicating that ppl exhibit its anti - atherosclerotic activity by blocking paf receptor . the specific antagonist of paf , bn 52021 , has been previously shown to exert beneficial effects against atherosclerosis development in rabbits . in the present study , atherogenic diet enriched in either oopl or ppl , which are specific paf antagonists , causes inhibition of early atherosclerosis development in rabbits despite the fact that paf levels are elevated in their blood . these findings suggest that paf - antagonists from olive oil and olive pomace are able to slow down the atherosclerotic process at least at its early stages by inhibiting paf binding and activity even in cases where paf levels are highly increased . in conclusion , olive pomace contains paf specific antagonists with similar bioactivities to those of olive oil . these bioactive microconstituents inhibit both specific paf binding and paf activity ( in vitro and in vivo ) , consequently inhibiting early atherosclerosis development . the above data reinforce the beneficial effect of food origin paf antagonists against atherosclerosis development and indicate a main role of paf in atherosclerosis .