the human atherosclerotic plaque is characterized by increased levels of oxidized lipoproteins , such as ldl , hdl , phospholipids , triglycerides [ 1 , 2 ] , oxidized cholesterol products ( oxysterols ) , ffas , and fatty acid derivatives , as well as proteins such as fibrinogen , apolipoprotein a - i ( apoa - i ) , clusterin , and paraoxonase ( pon ) [ 5 , 6 ] . accumulating cholesterol in the plaque tends to precipitate and crystallize , forming sharp edges with increased volume leading to rupture - prone fibrous caps . these  vulnerable plaques  have several features that differentiate them from their nonvulnerable counterparts : vulnerable plaques are less stable , characterized by a large lipid core , thin fibrous cap and less collagen , intraplaque hemorrhaging and infiltration of inflammatory cells without calcification . these unstable plaques are associated with increased ischemic events , and about 20% of ischemic strokes are attributed to carotid artery atherosclerosis . major serum biomarkers of plaque vulnerability are related to inflammatory and proteolytic markers , yet no biomarkers exist for regular clinical use to indicate high risk of neurological events and to select patients for carotid surgery . mammalian pons ( pon1 , pon2 , and pon3 ) are a unique family of calcium - dependent esterases / lactonases . this enzyme reduces macrophages ' cellular oxidative stress , decreases cholesterol - biosynthesis rate , and stimulates hdl - mediated macrophage cholesterol efflux , thus protecting against foam - cell formation and atherogenesis [ 9 , 10 ] . pon1-deficient mice are susceptible to the development of atherosclerosis , whereas overexpression of human pon1 in mice inhibits atherosclerosis development . epidemiological evidence demonstrates that low pon1 activity is associated with increased risk of cardiovascular disease . the hydrolytic lactonase , arylesterase , and paraoxonase activities of pon1 are all inactivated under oxidative stress . immunohistochemical analysis has shown accumulation of pon1 in the human lesion as it progresses from fatty streak to advanced lesion [ 5 , 16 ] . , pon2 has also been shown to protect vascular cells from oxidative stress , decrease triglyceride accumulation in macrophages [ 10 , 17 , 18 ] , play a role against inflammation , and exhibit high acylhomoserine lactone hydrolysis . pon3 , like pon1 , is associated with hdl but does not exhibit paraoxonase activity and is 200 times less abundant . we recently reported that lipids derived from carotid atherosclerotic plaque ( lesion lipids extract  lle ) can accelerate macrophage and lipoprotein oxidation and possess atherogenic properties , with the formation of macrophages with foam cell - like appearance . incubation of recombinant pon1 ( repon1 ) with lle reduces lipid carotid plaque atherogenicity [ 20 , 21 ] but at the same time , linoleic acid hydroperoxide ( la-13ooh ) present in the lle inhibits repon1 paraoxonase and lactonase activities via reaction of la-13ooh with the enzyme 's cysteine at position 284 ( cys284 ) . these dual effects between plaque constituents and the elements that are in contact with them , such as blood components ( lipids , proteins ) circulating via the lesion , led us to further investigate ( a ) if pons present in the human carotid plaque [ 16 , 23 ] are still active and ( b ) if differences between symptomatic and asymptomatic patients occur within the plaque constituents and between plaque and blood elements . n , o - bis(trimethylsilyl ) acetamide ( bsa ) , cholesterol , 2-hydroxyquinoline ( 2hq ) , diethyl p - nitrophenyl phosphate ( paraoxon ) , and 4-nitrophenyl acetate were purchased from sigma - aldrich . 7-hydroxycholesterol , 7-hydroxycholesterol , -epoxycholesterol , -epoxycholesterol , 26-hydroxycholesterol , and 7-ketocholesterol were purchased from steraloids inc . recombinant pon1 from escherichia coli was purchased from the structural proteomics center , weizmann institute of science ( rehovot , israel ) . human carotid plaques were taken from patients undergoing routine endarterectomy in the department of vascular surgery in carmel hospital ( haifa , israel ) . both symptomatic and asymptomatic patients were considered symptomatic if they had experienced stroke , transient ischemic attack , or amaurosis fugax ipsilateral to the carotid lesion being studied . complete atherosclerotic plaques were removed , including the common internal and external carotid sections and were immediately placed in saline and kept at 80c . all plaques were approved for research by the helsinki committee regulations , with patient consent ( helsinki approval number 3071 ) . lesion samples were laid on filter paper to absorb the liquid and then weighed and ground to a powder under liquid nitrogen . the powder was extracted at 4c for 30  min with pon activity buffer ( 1  mm cacl2 in 50  mm tris - hcl , ph 8.0 ) with 0.1% ( v / v ) protease inhibitor cocktail and 1  mm pmsf ( 1  ml for 400  mg tissue ) , and centrifuged at 10,000  g for 10  min at 4c . the supernatant was removed and used for determination of protein levels by dc protein assay ( bio - rad ) and for pon activities . ethyl acetate was evaporated , and the lle was dissolved in dmso to a final concentration of 50  mg / ml and used for detection of lipids and oxidized lipids by liquid chromatography - mass spectrometry ( lc - ms ) or gas chromatography ( gc - ms ) . pon1 storage buffer ( 50  mm tris , ph 8.0 , 50  mm nacl , 1  mm cacl2 , and 0.1% v / v tergitol ) was supplemented with 0.02% ( w / v ) sodium azide and stored at 4c . protein ( 40  g ) from the plaque homogenate , 0.1  g / ml repon1 , or 1  :  20 diluted serum was taken for a total reaction volume of 200  l . the assay mixture included 1  mm tbbl and 1  mm cacl2 in 50  mm tris - hcl , ph 8.0 . one unit of lactonase activity was equal to 1  mol of tbbl hydrolyzed / min ml . protein ( 100  g ) from the plaque homogenate or 0.2  g / ml repon1 was taken for a total reaction volume of 200  l . the basal assay mixture included 2  mm paraoxon and 1  mm cacl2 in 50  mm glycine / naoh buffer , ph 10.5 . one unit of pon1 paraoxonase activity was equal to 1  nmol of paraoxon hydrolyzed / min ml . protein ( 60  g ) from the plaque homogenate or 0.1  g / ml repon1 was taken for a total reaction volume of 200  l . the assay mixture included 3  mm 4-nitrophenyl acetate and 1  mm cacl2 in 50  mm tris - hcl , ph 8.0 . one unit of arylesterase activity was equal to 1  mol of 4-nitrophenyl acetate hydrolyzed / min ml . plaque triglyceride mass was determined using the serum triglyceride kit from sigma ( rehovot , israel ) . in short , plaque lipids dry samples were dissolved in dmso ( 50  mg / ml ) , and 50  l was added to 1  ml of glycerol reagent and 4  ml triglyceride reagent , vortexed and incubated for 10  min at 37c . samples were first subjected to hydrolysis to convert all sterol esters into their free form . the dry residue of the extracted sample was dissolved in 0.5  ml koh solution ( 20% koh in a mixture of meoh  :  ddw 70  :  30 ) and mixed for 3  h at 21c . two volumes of diethyl ether were added and the ph was adjusted to 5 with 0.5  ml of citric acid ( 20% in ddw ) . the upper organic phase was removed and the liquid phase was extracted with another portion of 2  ml diethyl ether . the organic layers were combined , treated with sodium sulfate , and evaporated to dryness under nitrogen purge . dried extracts were subjected to the silylating reagent bsa dissolved in 1,4-dioxane ( dried on 4  molecular sieves and passed through aluminum oxide ) and heated to 80c for 60  min . briefly , standards or dried extracts were subjected to 200  l bsa , followed by the addition of 300  l 1,4-dioxane ( treated as before ) and heated to 80c for 60  min . samples were detected by gc - ms in a total ion monitoring ( tim ) mode , and 24 of the most representative ions were selected for reinjection in single ion monitoring ( sim ) mode . ffas were quantified by lc - ms / hplc ( waters 2790 ) connected to an ms ( micromass quattro ultimams , uk ) . the hplc column was a 3.5  mm c18 ods xterra column ( waters ) . ms analysis of the ffas was performed in sim mode , using electron spray negative ions . ms / ms analysis of the oxidized products was performed in scan and daughter modes , using es . the source temperature of the ms was set at 120c , with a cone gas flow of 22  l / h and a desolvation gas flow of 400  l / h . collision - induced dissociation ms was performed , with a collision energy of 20  ev and 33.5  kv capillary voltage . multiple - reaction monitoring was performed under the same conditions used to quantify the oxidized products . statistical analysis was performed using the student paired or heteroscedastic t - test when comparing the means of two groups . each experiment was repeated , separately , at least three times ( n  3 ) . clinical parameters were analyzed using graphpad prism 4 software by contingency table with chi - square test . immunohistochemical analysis has recently revealed the presence of pon1 , pon3 , and pon2   in carotid lesions . however , such analyses do not indicate whether the enzymes in the plaque still possess hydrolytic activity . as part of our ongoing research into the chemical composition of the human carotid plaque , the mechanism by which plaque components interrelate , and their possible dual effects with blood components , we tested whether plaque pons are still active . the powder was extracted with tris buffer and then centrifuged ( see section 2 ) . the supernatant was used for determination of pon paraoxonase , lactonase and arylesterase activities , with or without the addition of the pon1 inhibitor 2hq . all three pon activities were preserved in the plaque homogenate , and 2hq almost completely inhibited paraoxonase and lactonase activities ( from 2.9 to 0.014 and from 0.115 to 0.008  u / mg protein , resp . , figures 1(a ) and 1(b ) ) , whereas it only slightly decreased arylesterase activity ( from 3.3 to 2.5  u / mg protein ) ( figure 1(c ) ) . 2hq inhibited repon1 paraoxonase , lactonase , and arylesterase activities almost completely ( from  9006  161  to 1819  30 ,  365  6  to 65.6  0.7 , and  118  14  to 9.9  2.8  u / mg repon1 protein , resp . ) these results indicated that pons that are present in the plaques preserve all three of their hydrolytic activities . atherosclerotic patients have been shown to have less pon1 activity in their blood than healthy subjects . since symptomatic patients have some features of vulnerable plaques , their plaques are considered to be more severe than those of asymptomatic patients [ 7 , 28 , 29 ] . in the present study , clinical parameters were compared between patients with hypertension , hyperlipidemia , or being treated with statins or antihypertensive drugs : no significant differences were found between symptomatic and asymptomatic patients in these criteria . the symptomatic group included more diabetic subjects than the asymptomatic group ( table 1 ) . surprisingly , there was no significant difference in plaque lactonase activity between the two groups ( table 2 ) . serum pon1 activity , as expected , was lower ( 15% ) in the symptomatic patients than in the asymptomatic patients , but the difference was not significant ( p = 0.14 ) ( table 2 ) . atherogenic components were then compared between symptomatic and asymptomatic patients , including levels of triglyceride , oxysterol , and la-13ooh in the plaque lipid extract , and levels of lipoproteins and hemoglobin a1c ( hba1c ) in individual patients ' blood . the levels of various lipids and oxidized lipids in the plaque extracts were determined by ms . no significant differences were found in the amounts of detected oxysterols ( 5,6-- and -epoxy cholesterol , 7-oh and 7-oh cholesterol , 7-keto cholesterol , and 26-oh cholesterol ) or triglycerides . in contrast , symptomatic patients had significantly higher ( 135% ) amounts of la-13ooh in their plaques than asymptomatic patients ( table 2 ) . in addition , while there was no significant difference in the amounts of total cholesterol , ldl cholesterol , or triglyceride in the blood , symptomatic patients had significantly less ( 28% ) hdl cholesterol than the asymptomatic group ( table 2 ) . symptomatic patients also had more ( 18% ) hba1c in their blood than asymptomatic patients , although this difference was only marginally significant ( table 2 ) . previous studies from our group have shown that la-13ooh ( with the hydroperoxide at position 13 of linoleic acid ) inhibits pon1 by specific interaction with the enzyme 's cys284 . thus , la-13ooh is considered an atherogenic factor in the plaque which can augment oxidative stress and progression of atherosclerosis . this led us to correlate la-13ooh level in the plaque with other atherogenic and antiatherogenic elements in the blood of the same patient , to assess the possible correlation between plaque status and a specific component in the blood . results showed that plaque la-13ooh level is indeed inversely correlated with two antiatherogenic elements in the blood : serum pon1 lactonase activity ( r = 0.35 , p = 0.01 , figure 2(a ) ) and hdl cholesterol ( r = 0.3 , p = 0.027 , figure 2(b ) ) . in addition , a direct correlation was found between the two atherogenic elements , la-13ooh and hba1c ( r = 0.27 , p = 0.038 , figure 2(c ) ) . the antioxidant enzyme pon1 lowers lipid peroxide levels , protects arterial cell walls ( as well as endothelial cells , smooth muscle cells , and macrophages ) and lipoproteins ( ldl and hdl ) from oxidation , inhibits oxidized ldl uptake by macrophages [ 9 , 10 ] , and shows anti - inflammatory activities [ 31 , 32 ] . thus , the presence of pon1 , pon2 , and pon3 in the human plaque may have an important role in decreasing atherosclerotic progression . immunohistochemical analysis of human plaque has revealed the presence of pons in the lesion ; during lesion progression , there is a shift of pon1 and pon3 from smooth muscle cells to macrophages , whereas arterial pon2 level decreases . hence , it is important to assess whether the pons identified immunohistochemically in the plaque are still active . our current results show that the pons are indeed still active in the plaque homogenate , preserving their catalytic paraoxonase , arylesterase , and lactonase activities . among these activities , only pon1 can hydrolyze paraoxon , hence we concluded that human carotid lesion has active pon1 . pon1 may thus act as a potent reducing antioxidant enzyme not only in the serum , but also within the plaque , leading to attenuated atherosclerotic progression . in addition , while 2hq inhibited repon1 arylesterase almost completely but only slightly decreased arylesterase in the homogenate , we can conclude that in the latter , other esterases are present and pon 's contribution is only minor . a distinction between symptomatic and asymptomatic patients through the identification of biomarkers could provide information on symptom occurrence . such biomarkers are not yet available but are needed to make appropriate decisions on the type of intervention required . previous studies from our group have shown that la-13ooh is present in the lipid extract of the human carotid plaque , and that it specifically inhibits repon1 activity in a dose- and time - dependent manner . during pon1 's interaction with lesion la-13ooh , the enzyme displayed a peroxidase - type of catalysis , reducing la-13ooh to la - oh ( hydroxide ) via the pon1 amino acid cys284 . thus , the levels of la-13ooh in symptomatic and asymptomatic patients were compared and correlated with antiatherogenic compounds in the plaque , or in the serum derived from these patients . symptomatic patients had significantly higher levels of la-13ooh in their plaques than their asymptomatic counterparts . in addition , la-13ooh levels in the plaque were significantly inversely correlated with serum pon1 activity ( figure 2(a ) ) and with serum hdl cholesterol ( figure 2(b ) ) . although pon1 is an hdl - associated enzyme , hdl particles are highly heterogeneous in their structure , intravascular metabolism , and biological activities . furthermore , pon1 is only present on a relatively small fraction of the hdl particles , mostly on the hdl3 subfraction . thus , the amount of the hdl in the blood is not necessarily correlated with serum pon1 activity . indeed , in this study , serum pon1 lactonase activity was not correlated with serum hdl levels ( figure 3 ) . these results indicate that la-13ooh might be affected , independently , by both serum pon1 and serum hdl level . as plaque la-13ooh has been shown to interact with and inhibit repon1 , and since pon1 is present and active , in both blood and the atherosclerotic plaque , we determined pon1 activity in serum and plaques derived from the same individuals and compared them in symptomatic versus asymptomatic patients . our results showed no differences in plaque pon activity between symptomatic and asymptomatic atherosclerotic patients ( p = 0.8 ) . serum pon1 activity in the symptomatic patients was lower than in the asymptomatic patients , although this difference was not significant ( p = 0.14 ) ( table 2 ) . plaque pon activity may be attributed to both pon1 and the intracellular enzyme pon2 , which may not be affected by the presence of la-13ooh . in addition , it has been previously shown that inhibition of repon1 by la-13ooh can be prevented if certain thiols , such as the amino acid cysteine , are present , and that pon1 inhibition by la-13ooh can be partially recovered if a thiol is added to the incubation system . la-13ooh probably oxidizes the pon1 cys284 to sulfenic acid , which can then be further oxidized to sulfinic and sulfonic acid derivatives . in the presence of thiol , the sulfenic acid derivative of cys284 can be reduced back to thiol ; however , if the oxidation proceeds further , addition of an external thiol can no longer reverse the reaction . we can , therefore , hypothesize that the presence of free cysteine in the blood and in the plaque may also prevent la-13ooh - induced pon1 inactivation .   in accordance with carr et al . , we did not observe significant differences in serum cholesterol or triglyceride levels between symptomatic and asymptomatic patients , or in triglyceride or oxysterol levels in the plaques themselves ( table 2 ) . in addition to their higher levels of plaque la-13ooh and lower levels of serum hdl relative to asymptomatic patients , there are more diabetics among the symptomatic patients ( table 1 ) , as characterized by a higher concentration of blood hba1c ( table 2 ) . hba1c is a glycated hemoglobin , which is highly prone to oxidation and its level is linked to oxidative stress . diabetic patients are a heterogeneous population that may differ in their exposure to risk factors . for example , haptoglobin ( hp ) genotype is an independent risk factor for vascular complications in diabetes . this study suggests la-13ooh as a possible risk factor for diabetic atherosclerosis development and complications , as analyses of plaque la-13ooh and blood hba1c revealed a direct correlation between these two atherogenic elements . levels of hba1c are strongly correlated with mean blood glucose , and high glucose concentration is known to increase oxidative stress . hba1c is also correlated with lipid peroxidation values , as shown in type 1 diabetic patients , as well as with plasma aldehydes ( malondialdehydes ) in type 2 diabetics [ 38 , 39 ] . furthermore , hussein et al .   recently showed that the lag time required for the initiation of ldl oxidation ( in the presence of copper ions ) is inversely correlated with hba1c concentration ( mainly when hba1c < 7.3% ) . moreover , incubation of red blood cell hemolysate with increasing concentrations of glucose and with ldl or oxidized ldl results in an increased concentration of hba1c . thus , the increased tendency for ldl to undergo lipid peroxidation in diabetic patients contributes to increased levels of blood hba1c . this further emphasizes the strong association between hba1c and oxidative stress , which is in agreement with the present findings pointing to a direct link between the atherogenic compounds in the atherosclerotic lesions and serum , such as la-13ooh and blood hba1c . in addition , it is in line with the inverse relationships shown for serum hdl and pon1 activity , which are both antiatherogenic elements of the blood .   in conclusion , la-13ooh levels in plaques from symptomatic patients are higher than those in asymptomatic patients ' plaques . the high levels of plaque la-13ooh are correlated with low levels of serum hdl , low levels of serum pon1 lactonase activity , and increased concentrations of blood hba1c , all of which further accelerate atherosclerotic progression . thus , the levels of pon1 activities , hdl concentration , and hba1c content could serve as suitable biomarkers to assess la-13ooh level in human carotid plaques .
human carotid plaque components interact directly with circulating blood elements and thus they might affect each other . 
 we determined plaque paraoxonase1 ( pon1 ) hydrolytic - catalytic activity and compared plaque and blood levels of lipids , hdl , pon1 , and hba1c , as well as plaque - oxidized lipids in symptomatic and asymptomatic patients . 
 human carotid plaques were obtained from symptomatic and asymptomatic patients undergoing routine endarterectomy , and the lesions were ground and extracted for pon activity and lipid content determinations . 
 plaque pons preserved paraoxonase , arylesterase , and lactonase activities . the pon1-specific inhibitor 2-hydroxyquinoline 
 almost completely inhibited paraoxonase and lactonase activities , while only moderately inhibiting arylesterase activity . 
 oxysterol and triglyceride levels in plaques from symptomatic and asymptomatic patients did not differ significantly , but plaques from symptomatic patients had significantly higher ( 135% ) linoleic acid hydroperoxide ( la-13ooh ) levels . their serum pon1 activity , cholesterol and triglyceride levels did not differ significantly , but symptomatic patients had significantly lower ( 28% ) serum hdl levels and higher ( 18% ) hba1c levels . 
 thus la-13ooh , a major atherogenic plaque element , showed significant negative correlations with serum pon1 activity and hdl levels , and a positive correlation with the prodiabetic atherogenic hba1c . 
 plaque pon1 retains its activity and may decrease plaque atherogenicity by reducing specific oxidized lipids ( e.g. , la-13ooh ) . the inverse correlation between plaque la-13ooh level and serum hdl level and pon1 activity suggests a role for serum hdl and pon1 in la-13ooh accumulation .