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Oxidative inactivation of paraoxonase—implications in diabetes mellitus and atherosclerosis
Biochimica et Biophysica Acta 1725 (2005) 213 – 221 http://www.elsevier.com/locate/bba
Oxidative inactivation of paraoxonase—Implications in diabetes mellitus and atherosclerosis Sonia-Athina P. Karabina a,1, Alexander N. Lehner b,1, Elizabeth Frank c, Sampath Parthasarathy d, Nalini Santanam d,* a INSERM U525 Faculte de Medecine Pitie-Salpetriere, Paris, France University College Northampton, Park Campus, Northamptonshire, England UK c Bio-Chem Foundation, Mysore, India d Department of Pathology, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112, USA b
Received 7 February 2005; received in revised form 29 June 2005; accepted 18 July 2005 Available online 3 August 2005
Abstract Human serum paraoxonase (PON1) has been implicated to play an important role in cardiovascular disease and diabetes. Studies in the literature indicate that PON1 has two different enzyme activities, i.e., esterase and hydroperoxide reducing activities. The objective of this study was to establish the importance of these two activities and to distinguish between them. As the addition of copper immediately inactivated the enzyme, we used auto-oxidation as the model system. Auto-oxidation of HDL resulted in more than 80% reduction of the esterolytic activity, which was protected by antioxidants, Vitamin E (50%) and PDTC (95%) and completely by 1 M glucose. In contrast, the hydroperoxide reducing activity, using unesterified hydroperoxides remained unaffected with time. We also used pNPHPODE (novel substrate) to establish that hydrolysis might be a prerequisite for the enzyme to act on the esterified hydroperoxide. The results indicated that the hydrolysis of the substrate was inhibited under oxidizing conditions with no reduction of the hydroperoxide. Overall, our findings suggest that protecting the esterolytic activity of PON1 by antioxidants might be important in preserving its action on phospholipid peroxides and a concerted reaction involving the esterolytic and hydroperoxide reducing activities might be suggested for the action of PON1. D 2005 Elsevier B.V. All rights reserved. Keywords: Lipid hydroperoxide; High density lipoprotein; Antioxidant; Glucose; Esterase; Low density lipoprotein
1. Introduction Human serum paraoxonase (PON1), a 43-kDa protein, catalyses the hydrolysis of organophosphate esters, aromatic carboxylic acid esters, and carbamates in a calcium dependent manner [1]. PON1 is synthesized in the liver and is mainly associated with high-density lipoprotein (HDL) [2]. Although the natural substrate for PON1 is still unknown, several groups reported that the enzyme protein has the capacity to retard the accumulation of lipid
* Corresponding author. Tel.: +1 504 568 2646; fax: +1 504 568 2932. E-mail address: [email protected] (N. Santanam). 1 Contributed equally to this study. 0304-4165/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2005.07.005
peroxides in low-density lipoprotein (LDL), and that this is mainly due to the ability of the enzyme to reduce hydroperoxides [3 – 5]. Aviram et al. showed that the esterolytic activity of PON1 in serum HDL correlated inversely with the susceptibility of HDL to oxidation [6] and that the active site involved in conferring protection to LDL oxidation is different than that involved in arylesterase/ paraoxonase activity [7]. However, the relationship between the esterolytic and hydroperoxide reducing activities is not fully understood. In addition, purified PON1 decreased the biological effects of mildly oxidized LDL and this was attributed to the ability of the enzyme to eliminate multi-oxygenated phospholipids produced during the oxidation of lipoproteins [8]. This was further supported when HDL isolated from PON1-
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deficient mice was unable to prevent oxidation of LDL in a co-cultured cell model of the artery wall. Both HDL and LDL isolated from PON1-knockout mice were equally susceptible to oxidation by co-cultured cells. Also, such mice developed significantly larger aortic lesions than the wild type littermates [9,10]. Recently, a phospholipase A2 activity was attributed to PON1 resulting in the release of lyso phosphatidylcholine that decreases the cholesterol biosynthesis is macrophages [11,12]. However, a recent study indicated that purified PON1 might lack hydroperoxide-reducing activity when tested using LDL and copper or AAPH as the oxidation system [13]. PON1 activity has been reported to be decreased in cardiovascular disease (CVD) [14,15], and in diabetes mellitus (DM) [3,16,17]. It was shown that in CVD, the activity and concentration of PON1 are more important than the PON1 genotype [18]. The inactivation of PON1 has been documented in literature [13,19]. Since oxidation is implicated in both CVD and DM, we speculate that the products of lipid peroxidation and glycation may directly modify PON1 protein. We show that (a) the hydroperoxide reducing activity is insensitive to chelating agents or oxidative inactivation, and (b) high glucose might actually protect the enzyme against oxidative inactivation. In addition, we provide evidence to suggest that the esterolytic activity might act in conjunction with the hydroperoxide reducing activity of the enzyme.
incubated with HDL in the absence and presence of glucose (1 M, 100 mM, or 5 mM) for 1 week at room temperature. Native HDL in PBS was kept for the same period of time at 4 -C. 2.4. Incubations with copper sulfate (CuSO4) Native HDL (100 Ag protein/ml) was mixed with 2.5– 20 AM CuSO4. Controls having the same volume of PBS were carried out to exclude the effect of dilution. PON1 activity in the HDL samples was measured immediately. 2.5. Measurement of enzymatic activities of PON1
2. Materials and methods
The esterolytic activity was measured using the rate of hydrolysis of paraoxon as assessed by the formation of pnitrophenol (pNP) at 405 nm at 25 -C [23]. Hydroperoxide reducing activity of PON1 was measured using a modified protocol of the N-benzoyl leucomethylene blue (LMB) assay developed by Auerbach et al. [24]. Serum, plasma (derived using different anticoagulants), or HDL (isolated from the serum or the various plasmas) were incubated with 13-hydroperoxyoctadecadienoic acid (13-HPODE) (250 AM) at 37 -C for 1 h. The 13HPODE was prepared as previously described [25]. After incubation with HDL, the remaining 13-HPODE was determined using the LMB assay. In control incubations, the decrease in LMB color intensity was linear with increasing duration of incubation of 13-HPODE with enzyme.
2.1. Sample collection
2.6. Western blot analysis
Samples were collected from healthy individuals and used fresh. The Human Investigations Committee of Emory University (Atlanta, GA) approved the human subject protocol. Plasma was excluded if Lipoprotein (a) was present or there were indications of dyslipidemia. Blood was collected in tubes containing either heparin, sodium citrate, EDTA, or no additive. LDL and HDL were isolated by density gradient ultracentrifugation [20].
Proteins were separated using SDS-PAGE [26]. Western blot was performed using rabbit anti-human PON1 (a generous gift from Dr. Srinivasa Reddy) at 1:1,000 and goat anti-human apoprotein A1 (ApoA1) (Rockland) at 1:5000 as primary antibodies. The proteins were detected by chemiluminescence using ECL detection kit (Amersham) according to the manufacturer’s instructions. The same set of samples was simultaneously loaded onto separate 12% SDS-PAGE gels and Coomassie stain was used to visualize the proteins.
2.2. Protein modification HDL was incubated in phosphate-buffered saline (PBS) containing 1 M, 100 mM, 5 mM glucose, or PBS alone for 1 week at room temperature. Native HDL in PBS was kept for the same period of time at 4 -C. Acetylated HDL and malondialdehyde (MDA)-HDL were prepared by methods previously described [21,22]. 2.3. Incubations with antioxidants Pyrrolidine dithiocarbamate (PDTC) (50 AM– 1 mM), Vitamin E (VitE) (50 AM –1 mM), Vitamin C (VitC) (50 AM– 1 mM), or N-acetylcysteine (50 AM– 1 mM) were
2.7. Synthesis of novel substrate precursor p-nitrophenyl linoleic acid (pNPLA) Linoleic anhydride (LA) was prepared by incubating 2 mmol linoleic acid with 1 mmol N,NVdicyclohexyl carbodiimide in 4 ml anhydrous carbon tetrachloride for 24 h. The precipitated dicyclohexyl urea was filtered off and the linoleic anhydride was obtained by evaporating the solvent [27]. LA was esterified to p-nitrophenyl (pNP) under anhydrous conditions in the presence of 10 Amol of dimethylaminopyridine which served as a catalyst [28]. The product, pNPLA was purified by silica
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gel column chromatography using increasing amounts of diethyl ether in hexane as solvent. The identity of the purified product was verified by the liberation of linoleic acid and p-NP upon hydrolysis and by thin layer chromatography (TLC). 2.8. Synthesis and characterization of p-nitrophenyl 13-hydroperoxylinoleate (pNPHPODE) Synthesis of the novel substrate, pNPHPODE: A twice purified ethanolic solution of pNPLA, devoid of free linoleic acid or pNP, was brought to room temperature and 1 Al of an ethanolic solution containing 100 nmol of pNPLA was added to 1 ml of PBS, vortexed for 1 s and transferred to a cuvette in a Uvikon spectrophotometer. After the addition of 10 Al Soybean lipoxygenase (SLO) (52,000 units/ml), the reaction was allowed to continue until no further increase in OD 234 was noticed even upon further addition of SLO. The presence of peroxide was established by the ability of the material to give a blue color with LMB reagent. Characterization of pNPHPODE: The pNPLA had no initial absorption at 234 nm or at 410 nm indicating the absence of conjugated dienes or free pNP in the starting material (Fig. 1A). Incubation with SLO resulted in the formation of conjugated dienes (Fig. 1B) with a peak around 234 nm, but no pNP was released as noted by the absence in absorption at 410 nm. Incubation of the oxidized product with LMB reagent revealed the formation of lipid peroxides (peak at 660 nm) and the presence of conjugated dienes (peak at 234 nm) (Fig. 1C). Hydrolysis of the product after lipoxygenase treatment with alkali showed both the peak for the production of peroxides (660 nm) and the release of free pNP at 410 nm (Fig. 1D). These results clearly established that lipoxygenase could act on the intact pNPLA and that the product was stable and did not undergo spontaneous hydrolysis during the incubation. It was also obvious that pNP was released only after the alkaline hydrolysis of the product.
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2.9. Verification of p-NPHPODE hydrolysis The enzymatic hydrolysis of pNPHPODE was performed in a 96-well plate. Substrates, (pNPA, pNPLA, or pNPHPODE) were diluted with ethanol and placed in the wells and then were allowed to air dry. 1 M Tris – HCl buffer at pH 7.4 and 50 Al plasma (prepared with either EDTA or Heparin) from the same subject were added. The plate was read in a microplate reader at 405 nm at repeated intervals. 2.10. Verification of reduction To determine the level of peroxidation, the p-NPHPODE was placed in a cuvette in the Uvikon spectrophotometer. A wavelength scan from 200 to 700 nm was performed (10 cycles) to assure the absence of any free pNP or benzene rings. Fifteen seconds prior to the second cycle, 300 Al LMB were added to the cuvette and mixed using a Pasteur pipette. The scan was then allowed to complete the remaining 9 cycles undisturbed. When determining the level of reduction, 80 Ag of HDL was mixed with 1 M Tris –HCl to a volume of 40 Al and were placed in three groups in a 96 well plate. As a control, a fourth group had only 40 Al of Tris buffer. The first group received 20 Al pNPHPODE. A half hour later, the second group received 20 Al pNPHPODE, and a half hour after that the Tris buffer group and the last HDL group received the pNPHPODE (simultaneously). All of the wells then received 100 Al of LMB and after incubation for 5 min were read in the plate reader at 620 nm. The measurement was repeated after 5 min to confirm there was no change in the relationship of the samples with time.
3. Results PON1 activity has been reported to be sensitive to calcium chelators and thus most studies employ serum for the measurement of the activity [29,30]. Many hydrolytic enzymes require Ca+2 for optimal activity. We could not however, rationalize the requirement of calcium for the
Fig. 1. Characterization of the novel substrate-pNPHPODE. pNPLA has no initial absorption at 234 nm or 404 nm (A). Addition of SLO results in pNPHPODE formation with peak at 238 nm (B). pNPHPODE + LMB reagent detects lipid peroxides (660 nm) (C). Hydrolysis of pNPHPODE with alkali showed both 660 and 410 nm peak (D).
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hydroperoxide reducing activity. Therefore, we determined both the esterolytic and the hydroperoxide reducing activities in plasma isolated with EDTA, citrate, or heparin. As noted in Fig. 2, the esterolytic activity that has been routinely measured as a measure of total PON1 activity, was severely inhibited by EDTA and citrate as opposed to the activity measured in heparinized plasma. The hydroperoxide reducing activity, however, was not affected by the presence of calcium chelators. It should be noted that the hydroperoxide activity was measured using free fatty acid hydroperoxides in these experiments. 3.1. Addition of copper to HDL has an instant inhibition of PON1 activity As mentioned earlier, PON1 activity is reduced in diabetic and atherosclerotic subjects, two diseases in which oxidation has been implicated. In order to determine whether the enzyme activities are susceptible to oxidative inactivation, we initially chose copper mediated oxidation of HDL. We and others have described that HDL could be oxidized by copper [31,32]. However, such protocols require considerable time requiring several hours in vitro to complete oxidation. Surprisingly, in our present studies, the mere addition of Cu2+ resulted in the immediate decrease in the esterolytic activity of PON1 in a concentration-dependent manner (data not shown). Our results with copper are in agreement with that observed earlier by Cao et al, who observed a similar decrease in PON activity but only after 2 h of incubation of HDL with copper [33]. Addition of 50 AM PDTC preserved the activity. PDTC however has a dual role as a metal chelating agent as well as that of antioxidant. In this case, it is unlikely that it acted as a metal chelator since use of other chelators, EDTA or citrate, resulted in a loss of esterolytic activity. It is possible that
PDTC chelated Cu2+ but had little or no effect on Ca2+, or more likely, paraoxonase activity is sensitive to oxidation even if there is little or no lipid oxidation. It has been recently shown that myeloperoxidase (MPO) product hypochlorous acid oxidizes HDL [34]. In order to determine if such oxidation inactivates the PON1 activity in our system, we incubated 100 Ag of HDL with 0.1 U MPO and 50 AM H2O2. However, we encountered a problem. H2O2 which is an absolute requirement for MPO and other peroxidases seemed to affect PON 1 activity even in the absence of MPO (data not shown). This could be due to a direct inhibition of PON 1 or due to the nature of H2O2 as a competitive substrate [6]. The results, however, offer yet another exciting possibility that increased PON1 could also protect against oxidation by MPO by depleting H 2O 2. 3.2. The esterolytic activity of PON1 is diminished by oxidation In order to test the susceptibility of PON1 to oxidation (as measured by the esterolytic activity), we submitted HDL to spontaneous oxidation. Incubation of HDL for 1 week at 4 -C preserved the esterolytic activity. In contrast, as shown in Fig. 3, the incubation of HDL at room temperature resulted in a progressive loss of activity over the same period. The presence of antioxidants prevented the loss of activity. However, the ability of Vitamin E to preserve activity was incomplete even at very high concentrations, whereas PDTC was able to completely preserve the esterolytic activity. These results indicate that although oxidation might be involved in inactivation of the esterolytic activity of PON1, it may not require lipid peroxidation. In other words, the inactivation could be due to a more watersoluble oxidant rather than lipid peroxy radicals. In contrast, when HDL was subjected to incubation at room temperature, there was no loss of the hydroperoxide reducing activity (data not shown). These results support the hypothesis that the two activities, although possibly performed by the same protein, might occur at different sites. 3.3. Esterolytic activity of PON1 in high glucose conditions
Fig. 2. Hydrolytic and lipid peroxide reducing activities of PON1. Hydrolytic activities (black columns) and lipid peroxide reducing activities (white columns) of PON1 were determined in the plasma as described in text. Values are expressed as mean T S.D. of separate trials.
Both paraoxonase activity and concentration are reduced in DM [3]. This might be related to the ability of glucose to generate advanced glycation end-products (AGE), or to initiate oxygen radical production. To our surprise, when we incubated HDL with very high concentrations of glucose (1 M), concentrations which are routinely used for generating advanced glycation end products, which is used to enhance AGE production in vitro [35,36], the esterolytic activity of PON1 was preserved (Fig. 3). This suggested that very high concentrations of glucose could have actually acted as an antioxidant, or the formation of AGE adducts via the modification of lysine residues preserved the activity. There
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Fig. 3. Esterolytic activity of PON1 treated with glucose (Glc). Paraoxonase activity of HDL samples incubated in the presence of glucose and/or antioxidants for 1 week as described in the text. Values are expressed as mean T S.D. of separate trials.
is precedence in literature for the ‘‘antioxidant’’ effects of glucose and other sugars [37]. For examples, molar concentrations of mannitol has long been used as a radical scavenger [38]. In contrast, when incubation of HDL was performed with physiologically normal (5 mM) or elevated glucose concentrations (up to 100 mM), there was a loss of the esterolytic activity of PON1. These data could suggest that lower concentrations of glucose may facilitate or sustain oxygen radical production whereas very high concentrations might be self-quenching. This was further supported by the fact that antioxidants (especially PDTC) were effective in protecting the esterolytic activity of PON1 similar to that seen with 1 M glucose. PDTC was also able to protect the esterolytic activity of PON1 even when the lower concentrations of glucose were employed, suggesting that the low concentrations of glucose were generating oxidants. This is supported by other studies which suggest glucose oxidation generates superoxide radicals and hydrogen peroxide [39] which would be quenched by aqueous antioxidants more efficiently than by lipid-soluble antioxidants.
glucose treatment to evaluate the possible effects of AGE formation, these results become extremely intriguing. From these results, it would appear that the conditions under which people experimentally initiate AGE formation (high molarity of glucose and lengthy incubation), which modifies lysines, protects the esterolytic activity. At the same time, our data clearly show paraoxonase is sensitive to just such modifications. There are at least two possible factors that need to be considered to resolve this apparent paradox. First, the degree of modification with MDA and acetylation under the conditions we used is very high. This method of acetylation has been suggested to derivatize over 80– 90% of lysine residues in proteins [40]. So these in vitro acetylations or incubations with aldehydes might suggest that extensive lipid peroxidation or lysine modifications would still be deleterious to the enzyme activity, whereas the amount of modification caused by AGE production may be insufficient. Second, is the fact that although we used a very high concentration of glucose, we incubated for only 1 week, whereas other investigators incubate anywhere from
3.4. Paraoxonase in HDL that has been modified by products of lipid peroxidation HDL lipids are vulnerable to peroxidation, and products of lipid peroxidation could interact with proteins in a number of different ways. For example, aldehydes such as MDA could form covalent links with the lysine residues of the protein. In order to determine whether products of lipid peroxidation would adversely affect the esterolytic activity, we used acetic anhydride or MDA to block lysine residues. These modifications, as seen in Fig. 4, were also able to decrease the esterolytic activity of PON1 as compared to native HDL. When considered with the previous data on
Fig. 4. Modification of lysines on PON1. Paraoxonase activity of HDL after lysine derivatization by MDA or acetylation is shown. Values are expressed as mean T S.D. of separate trials.
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weeks to months. Therefore, although high glucose was protective, with extensively longer incubations, enzymatic activity may have been lost due to lysine modifications via glycation. 3.5. Effect of antioxidants and glucose on immunoreactivity of PON1 Oxidation affects proteins in many ways including the hydrolysis of peptide bonds and by generating cross links. HDL stored at 4 -C gave a single immunoreactive band at ~43 kDa when probed with a polyclonal antibody against human PON1 (Fig. 5A—Lane 1). No other bands were immunostained under these conditions. Autoxidation of HDL resulted in a loss of this band with the generation of several other bands of both lower and higher molecular weight (Lane 2). Vitamin E, a lipophilic antioxidant inhibited the oxidation of lipids, but was not able to prevent the loss of immunoreactive PON1 (Lane 3). In contrast, PDTC which preserved the esterolytic activity, also maintained the stability of the protein (Lane 4). The immunoreactivity of PON1 in the presence of 1 mM PDTC was similar to that observed in the HDL kept for the same period of time at 4 -C. Vitamin C preserved neither the activity nor the immunoreactivity, and although N-acetylcysteine had a slight protective effect on the activity and immunoreactivity, the effectiveness did not vary when different concentrations were used (data not shown). Interestingly, high glucose concentrations prevented both the loss of activity and the loss of immunoreactive PON1 (Lanes 5 –7). Low glucose concentrations resulted in a complete loss of the ~43 kDa band. The addition of antioxidants, together with lower glucose concentrations appeared to have an additive effect in the preservation of intact PON1 immunoreactivity (Lanes
8 – 10). Thus, agents that protected the activity also prevented the loss of PON1 protein. Since PON1 is closely associated with HDL, we also determined the integrity of ApoA1 by western blot analysis in these experiments (Fig. 5B). Incubation of HDL at 25 -C resulted in the appearance of a lower band corresponding to Apo A1. Interestingly, the conditions that preserved both the immunoreactivity and esterolytic activity of PON1 also preserved the integrity of ApoA1, possibly suggesting a more general effect of protein oxidation under the experimental conditions. In addition, some of the high molecular weight forms of human PON1 bands also were immunostained with an antibody against ApoA1 suggesting possible covalent cross-linking between these two proteins. 3.6. Hydrolysis/Reduction of pNPHPODE by HDL We considered the possibility that the esterase activity of PON1 might be required for the hydroperoxide reducing activity. To test this possibility, we synthesized the long chain linoleic acid ester of pNP. First, we established the hydrolysis of long chain unoxidized pNP ester by HDL. Practically, there was no difference between the hydrolysis of pNP acetate and pNP linoleate suggesting that pNP linoleate could be used as the substrate for the esterolytic activity (data not shown). We then tested the hydrolysis of the oxidized form of pNPLA, namely pNPHPODE. The hydrolysis of pNPHPODE was measured using plasma isolated in the presence of heparin or EDTA. As expected, the heparinized plasma was very efficient in hydrolyzing the substrate whereas EDTA plasma was less effective (Fig. 6A). In contrast to the reduction of free fatty acid hydroperoxide, the ability of the plasma to reduce pNPHPODE paralleled the hydrolytic
Fig. 5. Western blot analysis. Detection of PON1 using antibody to human PON1 (A) and ApoA1 using antibody to human ApoA1 (B) after incubation of HDL with additives for 1 week. The black arrow points to PON1 at ¨43 kDa, and the white arrow points to ApoA1 at ¨28 kDa.
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Fig. 6. The hydrolysis of pNPHPODE precursor (pNPLA) and reduction of pNPHPODE. The hydrolysis of pNPLa is comparable to phenyl acetate (pNPA) using heparinized and EDTA plasma (A). Reduction of pNPHPODE using heparinized and EDTA plasma (B). Data represented as mean T S.D.
activity indicating that hydrolysis was required for the reduction of the esterified fatty acid hydroperoxide (Fig. 6B). As could be expected, a non-enzymatic alkaline hydrolysis prior to incubation with the plasma that released the free fatty acid hydroperoxide showed no distinction between EDTA and heparin plasma.
4. Discussion The protective role of HDL in atherosclerosis has been well established and supported by clinical and epidemiological data. Apart from the reverse cholesterol transport, HDL also carries several anti-inflammatory enzymes. Among them, the role of lecithin cholesterol acyl transferase (LCAT), platelet activating factor acetylhydrolase (PAFAH) and paraoxonase (PON 1) has been studied extensively in atherosclerosis. Although these enzymes can act on similar substrates, they can still be distinguished from each other. Several years ago Subramanian et al. showed that, while PAF-AH carries out the hydrolysis of mainly truncated oxidized phosphatidylcholines, LCAT hydrolyzes
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and transesterifies the long-chain oxidized phosphatidylcholines [41]. In our experiments, we used whole HDL instead of purified PON, to show the effect of esterolytic and hydroperoxide reducing activities ascribed to PON 1, as it has been shown that both lipid and protein components of HDL are important for achieving optimal activity of the enzyme [42]. Additionally, experiments with purified PON 1 would also be deficient in other proteins and non-protein components present in HDL that may be essential for its physiological role in vivo [13]. Based on our data, we propose that there could be two different active sites in PON1 each specific for the estrolytic and hydroperoxide reducing activities and also that these activities differ in their mechanism of action. The implications of this finding are noteworthy. It is possible that in vivo these two activities work pari passu, wherein the hydroperoxide activity uses the product of the esterolytic activity as a substrate almost simultaneously as it is produced. As noted, the fatty acid peroxide reducing activity was not affected by autooxidation. However, the enzyme protein undergoes considerable changes under these conditions. Both fragmentation as well as ‘‘aggregation’’ has been noted. This would suggest that if intact lipid peroxides are the preferred substrate for the enzyme, then the sensitivity of the enzyme (i.e., the esterase activity) to oxidative stress might not be an important factor for consideration. On the other hand, if the esterolytic activities generate the preferred substrate, i.e., free fatty acid hydroperoxide, then it is important to preserve the esterolytic activity by use of antioxidants. Currently, paraoxon and aryl esters are used as substrates for PON1. There are no long chain esters of pNP or other aromatic compounds in circulation. It has been suggested that oxidized LDL phospholipids and cholesteryl esters are physiological substrates for serum paraoxonase [43]. One needs to wonder the need for such an esterase as several other enzymes, for example LCAT, PAF-acetyl hydrolase, and others could perform similar functions. Additionally, it has been shown that paraoxonase is able to reduce hydrogen peroxide (H2O2), a major reactive species produced during oxidative stress [6]. Considering the universal ‘‘acceptance’’ of esterase activity as a correlative parameter with atherosclerosis, it is tempting to speculate a concerted mechanism for the action of PON1 on the reduction of lipid hydroperoxides that would involve a hydrolysis of the esterified lipid to free fatty acid hydroperoxide followed by its reduction. If so, by preventing the oxidative inactivation of the esterolytic activity of PON1, suitable antioxidants would enhance the enzyme’s action on the detoxification of esterified lipid hydroperoxides. Considering that fat-soluble antioxidant vitamin E showed only a partial protection, whereas the water-soluble antioxidant vitamin C afforded little or no protection, there is a need for testing other water-soluble antioxidants.
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Acknowledgements This work was supported by NIH grant HL-069038 and DK-56353. We are grateful to Dr. Srinivasa Reddy, UCLA, for his generous gift of the antibody to PON1.
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Oxidative inactivation of paraoxonase—Implications in diabetes mellitus and atherosclerosis Sonia-Athina P. Karabina a,1, Alexander N. Lehner b,1, Elizabeth Frank c, Sampath Parthasarathy d, Nalini Santanam d,* a INSERM U525 Faculte de Medecine Pitie-Salpetriere, Paris, France University College Northampton, Park Campus, Northamptonshire, England UK c Bio-Chem Foundation, Mysore, India d Department of Pathology, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112, USA b
Received 7 February 2005; received in revised form 29 June 2005; accepted 18 July 2005 Available online 3 August 2005
Abstract Human serum paraoxonase (PON1) has been implicated to play an important role in cardiovascular disease and diabetes. Studies in the literature indicate that PON1 has two different enzyme activities, i.e., esterase and hydroperoxide reducing activities. The objective of this study was to establish the importance of these two activities and to distinguish between them. As the addition of copper immediately inactivated the enzyme, we used auto-oxidation as the model system. Auto-oxidation of HDL resulted in more than 80% reduction of the esterolytic activity, which was protected by antioxidants, Vitamin E (50%) and PDTC (95%) and completely by 1 M glucose. In contrast, the hydroperoxide reducing activity, using unesterified hydroperoxides remained unaffected with time. We also used pNPHPODE (novel substrate) to establish that hydrolysis might be a prerequisite for the enzyme to act on the esterified hydroperoxide. The results indicated that the hydrolysis of the substrate was inhibited under oxidizing conditions with no reduction of the hydroperoxide. Overall, our findings suggest that protecting the esterolytic activity of PON1 by antioxidants might be important in preserving its action on phospholipid peroxides and a concerted reaction involving the esterolytic and hydroperoxide reducing activities might be suggested for the action of PON1. D 2005 Elsevier B.V. All rights reserved. Keywords: Lipid hydroperoxide; High density lipoprotein; Antioxidant; Glucose; Esterase; Low density lipoprotein
1. Introduction Human serum paraoxonase (PON1), a 43-kDa protein, catalyses the hydrolysis of organophosphate esters, aromatic carboxylic acid esters, and carbamates in a calcium dependent manner [1]. PON1 is synthesized in the liver and is mainly associated with high-density lipoprotein (HDL) [2]. Although the natural substrate for PON1 is still unknown, several groups reported that the enzyme protein has the capacity to retard the accumulation of lipid
* Corresponding author. Tel.: +1 504 568 2646; fax: +1 504 568 2932. E-mail address: [email protected] (N. Santanam). 1 Contributed equally to this study. 0304-4165/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2005.07.005
peroxides in low-density lipoprotein (LDL), and that this is mainly due to the ability of the enzyme to reduce hydroperoxides [3 – 5]. Aviram et al. showed that the esterolytic activity of PON1 in serum HDL correlated inversely with the susceptibility of HDL to oxidation [6] and that the active site involved in conferring protection to LDL oxidation is different than that involved in arylesterase/ paraoxonase activity [7]. However, the relationship between the esterolytic and hydroperoxide reducing activities is not fully understood. In addition, purified PON1 decreased the biological effects of mildly oxidized LDL and this was attributed to the ability of the enzyme to eliminate multi-oxygenated phospholipids produced during the oxidation of lipoproteins [8]. This was further supported when HDL isolated from PON1-
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deficient mice was unable to prevent oxidation of LDL in a co-cultured cell model of the artery wall. Both HDL and LDL isolated from PON1-knockout mice were equally susceptible to oxidation by co-cultured cells. Also, such mice developed significantly larger aortic lesions than the wild type littermates [9,10]. Recently, a phospholipase A2 activity was attributed to PON1 resulting in the release of lyso phosphatidylcholine that decreases the cholesterol biosynthesis is macrophages [11,12]. However, a recent study indicated that purified PON1 might lack hydroperoxide-reducing activity when tested using LDL and copper or AAPH as the oxidation system [13]. PON1 activity has been reported to be decreased in cardiovascular disease (CVD) [14,15], and in diabetes mellitus (DM) [3,16,17]. It was shown that in CVD, the activity and concentration of PON1 are more important than the PON1 genotype [18]. The inactivation of PON1 has been documented in literature [13,19]. Since oxidation is implicated in both CVD and DM, we speculate that the products of lipid peroxidation and glycation may directly modify PON1 protein. We show that (a) the hydroperoxide reducing activity is insensitive to chelating agents or oxidative inactivation, and (b) high glucose might actually protect the enzyme against oxidative inactivation. In addition, we provide evidence to suggest that the esterolytic activity might act in conjunction with the hydroperoxide reducing activity of the enzyme.
incubated with HDL in the absence and presence of glucose (1 M, 100 mM, or 5 mM) for 1 week at room temperature. Native HDL in PBS was kept for the same period of time at 4 -C. 2.4. Incubations with copper sulfate (CuSO4) Native HDL (100 Ag protein/ml) was mixed with 2.5– 20 AM CuSO4. Controls having the same volume of PBS were carried out to exclude the effect of dilution. PON1 activity in the HDL samples was measured immediately. 2.5. Measurement of enzymatic activities of PON1
2. Materials and methods
The esterolytic activity was measured using the rate of hydrolysis of paraoxon as assessed by the formation of pnitrophenol (pNP) at 405 nm at 25 -C [23]. Hydroperoxide reducing activity of PON1 was measured using a modified protocol of the N-benzoyl leucomethylene blue (LMB) assay developed by Auerbach et al. [24]. Serum, plasma (derived using different anticoagulants), or HDL (isolated from the serum or the various plasmas) were incubated with 13-hydroperoxyoctadecadienoic acid (13-HPODE) (250 AM) at 37 -C for 1 h. The 13HPODE was prepared as previously described [25]. After incubation with HDL, the remaining 13-HPODE was determined using the LMB assay. In control incubations, the decrease in LMB color intensity was linear with increasing duration of incubation of 13-HPODE with enzyme.
2.1. Sample collection
2.6. Western blot analysis
Samples were collected from healthy individuals and used fresh. The Human Investigations Committee of Emory University (Atlanta, GA) approved the human subject protocol. Plasma was excluded if Lipoprotein (a) was present or there were indications of dyslipidemia. Blood was collected in tubes containing either heparin, sodium citrate, EDTA, or no additive. LDL and HDL were isolated by density gradient ultracentrifugation [20].
Proteins were separated using SDS-PAGE [26]. Western blot was performed using rabbit anti-human PON1 (a generous gift from Dr. Srinivasa Reddy) at 1:1,000 and goat anti-human apoprotein A1 (ApoA1) (Rockland) at 1:5000 as primary antibodies. The proteins were detected by chemiluminescence using ECL detection kit (Amersham) according to the manufacturer’s instructions. The same set of samples was simultaneously loaded onto separate 12% SDS-PAGE gels and Coomassie stain was used to visualize the proteins.
2.2. Protein modification HDL was incubated in phosphate-buffered saline (PBS) containing 1 M, 100 mM, 5 mM glucose, or PBS alone for 1 week at room temperature. Native HDL in PBS was kept for the same period of time at 4 -C. Acetylated HDL and malondialdehyde (MDA)-HDL were prepared by methods previously described [21,22]. 2.3. Incubations with antioxidants Pyrrolidine dithiocarbamate (PDTC) (50 AM– 1 mM), Vitamin E (VitE) (50 AM –1 mM), Vitamin C (VitC) (50 AM– 1 mM), or N-acetylcysteine (50 AM– 1 mM) were
2.7. Synthesis of novel substrate precursor p-nitrophenyl linoleic acid (pNPLA) Linoleic anhydride (LA) was prepared by incubating 2 mmol linoleic acid with 1 mmol N,NVdicyclohexyl carbodiimide in 4 ml anhydrous carbon tetrachloride for 24 h. The precipitated dicyclohexyl urea was filtered off and the linoleic anhydride was obtained by evaporating the solvent [27]. LA was esterified to p-nitrophenyl (pNP) under anhydrous conditions in the presence of 10 Amol of dimethylaminopyridine which served as a catalyst [28]. The product, pNPLA was purified by silica
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gel column chromatography using increasing amounts of diethyl ether in hexane as solvent. The identity of the purified product was verified by the liberation of linoleic acid and p-NP upon hydrolysis and by thin layer chromatography (TLC). 2.8. Synthesis and characterization of p-nitrophenyl 13-hydroperoxylinoleate (pNPHPODE) Synthesis of the novel substrate, pNPHPODE: A twice purified ethanolic solution of pNPLA, devoid of free linoleic acid or pNP, was brought to room temperature and 1 Al of an ethanolic solution containing 100 nmol of pNPLA was added to 1 ml of PBS, vortexed for 1 s and transferred to a cuvette in a Uvikon spectrophotometer. After the addition of 10 Al Soybean lipoxygenase (SLO) (52,000 units/ml), the reaction was allowed to continue until no further increase in OD 234 was noticed even upon further addition of SLO. The presence of peroxide was established by the ability of the material to give a blue color with LMB reagent. Characterization of pNPHPODE: The pNPLA had no initial absorption at 234 nm or at 410 nm indicating the absence of conjugated dienes or free pNP in the starting material (Fig. 1A). Incubation with SLO resulted in the formation of conjugated dienes (Fig. 1B) with a peak around 234 nm, but no pNP was released as noted by the absence in absorption at 410 nm. Incubation of the oxidized product with LMB reagent revealed the formation of lipid peroxides (peak at 660 nm) and the presence of conjugated dienes (peak at 234 nm) (Fig. 1C). Hydrolysis of the product after lipoxygenase treatment with alkali showed both the peak for the production of peroxides (660 nm) and the release of free pNP at 410 nm (Fig. 1D). These results clearly established that lipoxygenase could act on the intact pNPLA and that the product was stable and did not undergo spontaneous hydrolysis during the incubation. It was also obvious that pNP was released only after the alkaline hydrolysis of the product.
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2.9. Verification of p-NPHPODE hydrolysis The enzymatic hydrolysis of pNPHPODE was performed in a 96-well plate. Substrates, (pNPA, pNPLA, or pNPHPODE) were diluted with ethanol and placed in the wells and then were allowed to air dry. 1 M Tris – HCl buffer at pH 7.4 and 50 Al plasma (prepared with either EDTA or Heparin) from the same subject were added. The plate was read in a microplate reader at 405 nm at repeated intervals. 2.10. Verification of reduction To determine the level of peroxidation, the p-NPHPODE was placed in a cuvette in the Uvikon spectrophotometer. A wavelength scan from 200 to 700 nm was performed (10 cycles) to assure the absence of any free pNP or benzene rings. Fifteen seconds prior to the second cycle, 300 Al LMB were added to the cuvette and mixed using a Pasteur pipette. The scan was then allowed to complete the remaining 9 cycles undisturbed. When determining the level of reduction, 80 Ag of HDL was mixed with 1 M Tris –HCl to a volume of 40 Al and were placed in three groups in a 96 well plate. As a control, a fourth group had only 40 Al of Tris buffer. The first group received 20 Al pNPHPODE. A half hour later, the second group received 20 Al pNPHPODE, and a half hour after that the Tris buffer group and the last HDL group received the pNPHPODE (simultaneously). All of the wells then received 100 Al of LMB and after incubation for 5 min were read in the plate reader at 620 nm. The measurement was repeated after 5 min to confirm there was no change in the relationship of the samples with time.
3. Results PON1 activity has been reported to be sensitive to calcium chelators and thus most studies employ serum for the measurement of the activity [29,30]. Many hydrolytic enzymes require Ca+2 for optimal activity. We could not however, rationalize the requirement of calcium for the
Fig. 1. Characterization of the novel substrate-pNPHPODE. pNPLA has no initial absorption at 234 nm or 404 nm (A). Addition of SLO results in pNPHPODE formation with peak at 238 nm (B). pNPHPODE + LMB reagent detects lipid peroxides (660 nm) (C). Hydrolysis of pNPHPODE with alkali showed both 660 and 410 nm peak (D).
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hydroperoxide reducing activity. Therefore, we determined both the esterolytic and the hydroperoxide reducing activities in plasma isolated with EDTA, citrate, or heparin. As noted in Fig. 2, the esterolytic activity that has been routinely measured as a measure of total PON1 activity, was severely inhibited by EDTA and citrate as opposed to the activity measured in heparinized plasma. The hydroperoxide reducing activity, however, was not affected by the presence of calcium chelators. It should be noted that the hydroperoxide activity was measured using free fatty acid hydroperoxides in these experiments. 3.1. Addition of copper to HDL has an instant inhibition of PON1 activity As mentioned earlier, PON1 activity is reduced in diabetic and atherosclerotic subjects, two diseases in which oxidation has been implicated. In order to determine whether the enzyme activities are susceptible to oxidative inactivation, we initially chose copper mediated oxidation of HDL. We and others have described that HDL could be oxidized by copper [31,32]. However, such protocols require considerable time requiring several hours in vitro to complete oxidation. Surprisingly, in our present studies, the mere addition of Cu2+ resulted in the immediate decrease in the esterolytic activity of PON1 in a concentration-dependent manner (data not shown). Our results with copper are in agreement with that observed earlier by Cao et al, who observed a similar decrease in PON activity but only after 2 h of incubation of HDL with copper [33]. Addition of 50 AM PDTC preserved the activity. PDTC however has a dual role as a metal chelating agent as well as that of antioxidant. In this case, it is unlikely that it acted as a metal chelator since use of other chelators, EDTA or citrate, resulted in a loss of esterolytic activity. It is possible that
PDTC chelated Cu2+ but had little or no effect on Ca2+, or more likely, paraoxonase activity is sensitive to oxidation even if there is little or no lipid oxidation. It has been recently shown that myeloperoxidase (MPO) product hypochlorous acid oxidizes HDL [34]. In order to determine if such oxidation inactivates the PON1 activity in our system, we incubated 100 Ag of HDL with 0.1 U MPO and 50 AM H2O2. However, we encountered a problem. H2O2 which is an absolute requirement for MPO and other peroxidases seemed to affect PON 1 activity even in the absence of MPO (data not shown). This could be due to a direct inhibition of PON 1 or due to the nature of H2O2 as a competitive substrate [6]. The results, however, offer yet another exciting possibility that increased PON1 could also protect against oxidation by MPO by depleting H 2O 2. 3.2. The esterolytic activity of PON1 is diminished by oxidation In order to test the susceptibility of PON1 to oxidation (as measured by the esterolytic activity), we submitted HDL to spontaneous oxidation. Incubation of HDL for 1 week at 4 -C preserved the esterolytic activity. In contrast, as shown in Fig. 3, the incubation of HDL at room temperature resulted in a progressive loss of activity over the same period. The presence of antioxidants prevented the loss of activity. However, the ability of Vitamin E to preserve activity was incomplete even at very high concentrations, whereas PDTC was able to completely preserve the esterolytic activity. These results indicate that although oxidation might be involved in inactivation of the esterolytic activity of PON1, it may not require lipid peroxidation. In other words, the inactivation could be due to a more watersoluble oxidant rather than lipid peroxy radicals. In contrast, when HDL was subjected to incubation at room temperature, there was no loss of the hydroperoxide reducing activity (data not shown). These results support the hypothesis that the two activities, although possibly performed by the same protein, might occur at different sites. 3.3. Esterolytic activity of PON1 in high glucose conditions
Fig. 2. Hydrolytic and lipid peroxide reducing activities of PON1. Hydrolytic activities (black columns) and lipid peroxide reducing activities (white columns) of PON1 were determined in the plasma as described in text. Values are expressed as mean T S.D. of separate trials.
Both paraoxonase activity and concentration are reduced in DM [3]. This might be related to the ability of glucose to generate advanced glycation end-products (AGE), or to initiate oxygen radical production. To our surprise, when we incubated HDL with very high concentrations of glucose (1 M), concentrations which are routinely used for generating advanced glycation end products, which is used to enhance AGE production in vitro [35,36], the esterolytic activity of PON1 was preserved (Fig. 3). This suggested that very high concentrations of glucose could have actually acted as an antioxidant, or the formation of AGE adducts via the modification of lysine residues preserved the activity. There
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Fig. 3. Esterolytic activity of PON1 treated with glucose (Glc). Paraoxonase activity of HDL samples incubated in the presence of glucose and/or antioxidants for 1 week as described in the text. Values are expressed as mean T S.D. of separate trials.
is precedence in literature for the ‘‘antioxidant’’ effects of glucose and other sugars [37]. For examples, molar concentrations of mannitol has long been used as a radical scavenger [38]. In contrast, when incubation of HDL was performed with physiologically normal (5 mM) or elevated glucose concentrations (up to 100 mM), there was a loss of the esterolytic activity of PON1. These data could suggest that lower concentrations of glucose may facilitate or sustain oxygen radical production whereas very high concentrations might be self-quenching. This was further supported by the fact that antioxidants (especially PDTC) were effective in protecting the esterolytic activity of PON1 similar to that seen with 1 M glucose. PDTC was also able to protect the esterolytic activity of PON1 even when the lower concentrations of glucose were employed, suggesting that the low concentrations of glucose were generating oxidants. This is supported by other studies which suggest glucose oxidation generates superoxide radicals and hydrogen peroxide [39] which would be quenched by aqueous antioxidants more efficiently than by lipid-soluble antioxidants.
glucose treatment to evaluate the possible effects of AGE formation, these results become extremely intriguing. From these results, it would appear that the conditions under which people experimentally initiate AGE formation (high molarity of glucose and lengthy incubation), which modifies lysines, protects the esterolytic activity. At the same time, our data clearly show paraoxonase is sensitive to just such modifications. There are at least two possible factors that need to be considered to resolve this apparent paradox. First, the degree of modification with MDA and acetylation under the conditions we used is very high. This method of acetylation has been suggested to derivatize over 80– 90% of lysine residues in proteins [40]. So these in vitro acetylations or incubations with aldehydes might suggest that extensive lipid peroxidation or lysine modifications would still be deleterious to the enzyme activity, whereas the amount of modification caused by AGE production may be insufficient. Second, is the fact that although we used a very high concentration of glucose, we incubated for only 1 week, whereas other investigators incubate anywhere from
3.4. Paraoxonase in HDL that has been modified by products of lipid peroxidation HDL lipids are vulnerable to peroxidation, and products of lipid peroxidation could interact with proteins in a number of different ways. For example, aldehydes such as MDA could form covalent links with the lysine residues of the protein. In order to determine whether products of lipid peroxidation would adversely affect the esterolytic activity, we used acetic anhydride or MDA to block lysine residues. These modifications, as seen in Fig. 4, were also able to decrease the esterolytic activity of PON1 as compared to native HDL. When considered with the previous data on
Fig. 4. Modification of lysines on PON1. Paraoxonase activity of HDL after lysine derivatization by MDA or acetylation is shown. Values are expressed as mean T S.D. of separate trials.
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weeks to months. Therefore, although high glucose was protective, with extensively longer incubations, enzymatic activity may have been lost due to lysine modifications via glycation. 3.5. Effect of antioxidants and glucose on immunoreactivity of PON1 Oxidation affects proteins in many ways including the hydrolysis of peptide bonds and by generating cross links. HDL stored at 4 -C gave a single immunoreactive band at ~43 kDa when probed with a polyclonal antibody against human PON1 (Fig. 5A—Lane 1). No other bands were immunostained under these conditions. Autoxidation of HDL resulted in a loss of this band with the generation of several other bands of both lower and higher molecular weight (Lane 2). Vitamin E, a lipophilic antioxidant inhibited the oxidation of lipids, but was not able to prevent the loss of immunoreactive PON1 (Lane 3). In contrast, PDTC which preserved the esterolytic activity, also maintained the stability of the protein (Lane 4). The immunoreactivity of PON1 in the presence of 1 mM PDTC was similar to that observed in the HDL kept for the same period of time at 4 -C. Vitamin C preserved neither the activity nor the immunoreactivity, and although N-acetylcysteine had a slight protective effect on the activity and immunoreactivity, the effectiveness did not vary when different concentrations were used (data not shown). Interestingly, high glucose concentrations prevented both the loss of activity and the loss of immunoreactive PON1 (Lanes 5 –7). Low glucose concentrations resulted in a complete loss of the ~43 kDa band. The addition of antioxidants, together with lower glucose concentrations appeared to have an additive effect in the preservation of intact PON1 immunoreactivity (Lanes
8 – 10). Thus, agents that protected the activity also prevented the loss of PON1 protein. Since PON1 is closely associated with HDL, we also determined the integrity of ApoA1 by western blot analysis in these experiments (Fig. 5B). Incubation of HDL at 25 -C resulted in the appearance of a lower band corresponding to Apo A1. Interestingly, the conditions that preserved both the immunoreactivity and esterolytic activity of PON1 also preserved the integrity of ApoA1, possibly suggesting a more general effect of protein oxidation under the experimental conditions. In addition, some of the high molecular weight forms of human PON1 bands also were immunostained with an antibody against ApoA1 suggesting possible covalent cross-linking between these two proteins. 3.6. Hydrolysis/Reduction of pNPHPODE by HDL We considered the possibility that the esterase activity of PON1 might be required for the hydroperoxide reducing activity. To test this possibility, we synthesized the long chain linoleic acid ester of pNP. First, we established the hydrolysis of long chain unoxidized pNP ester by HDL. Practically, there was no difference between the hydrolysis of pNP acetate and pNP linoleate suggesting that pNP linoleate could be used as the substrate for the esterolytic activity (data not shown). We then tested the hydrolysis of the oxidized form of pNPLA, namely pNPHPODE. The hydrolysis of pNPHPODE was measured using plasma isolated in the presence of heparin or EDTA. As expected, the heparinized plasma was very efficient in hydrolyzing the substrate whereas EDTA plasma was less effective (Fig. 6A). In contrast to the reduction of free fatty acid hydroperoxide, the ability of the plasma to reduce pNPHPODE paralleled the hydrolytic
Fig. 5. Western blot analysis. Detection of PON1 using antibody to human PON1 (A) and ApoA1 using antibody to human ApoA1 (B) after incubation of HDL with additives for 1 week. The black arrow points to PON1 at ¨43 kDa, and the white arrow points to ApoA1 at ¨28 kDa.
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Fig. 6. The hydrolysis of pNPHPODE precursor (pNPLA) and reduction of pNPHPODE. The hydrolysis of pNPLa is comparable to phenyl acetate (pNPA) using heparinized and EDTA plasma (A). Reduction of pNPHPODE using heparinized and EDTA plasma (B). Data represented as mean T S.D.
activity indicating that hydrolysis was required for the reduction of the esterified fatty acid hydroperoxide (Fig. 6B). As could be expected, a non-enzymatic alkaline hydrolysis prior to incubation with the plasma that released the free fatty acid hydroperoxide showed no distinction between EDTA and heparin plasma.
4. Discussion The protective role of HDL in atherosclerosis has been well established and supported by clinical and epidemiological data. Apart from the reverse cholesterol transport, HDL also carries several anti-inflammatory enzymes. Among them, the role of lecithin cholesterol acyl transferase (LCAT), platelet activating factor acetylhydrolase (PAFAH) and paraoxonase (PON 1) has been studied extensively in atherosclerosis. Although these enzymes can act on similar substrates, they can still be distinguished from each other. Several years ago Subramanian et al. showed that, while PAF-AH carries out the hydrolysis of mainly truncated oxidized phosphatidylcholines, LCAT hydrolyzes
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and transesterifies the long-chain oxidized phosphatidylcholines [41]. In our experiments, we used whole HDL instead of purified PON, to show the effect of esterolytic and hydroperoxide reducing activities ascribed to PON 1, as it has been shown that both lipid and protein components of HDL are important for achieving optimal activity of the enzyme [42]. Additionally, experiments with purified PON 1 would also be deficient in other proteins and non-protein components present in HDL that may be essential for its physiological role in vivo [13]. Based on our data, we propose that there could be two different active sites in PON1 each specific for the estrolytic and hydroperoxide reducing activities and also that these activities differ in their mechanism of action. The implications of this finding are noteworthy. It is possible that in vivo these two activities work pari passu, wherein the hydroperoxide activity uses the product of the esterolytic activity as a substrate almost simultaneously as it is produced. As noted, the fatty acid peroxide reducing activity was not affected by autooxidation. However, the enzyme protein undergoes considerable changes under these conditions. Both fragmentation as well as ‘‘aggregation’’ has been noted. This would suggest that if intact lipid peroxides are the preferred substrate for the enzyme, then the sensitivity of the enzyme (i.e., the esterase activity) to oxidative stress might not be an important factor for consideration. On the other hand, if the esterolytic activities generate the preferred substrate, i.e., free fatty acid hydroperoxide, then it is important to preserve the esterolytic activity by use of antioxidants. Currently, paraoxon and aryl esters are used as substrates for PON1. There are no long chain esters of pNP or other aromatic compounds in circulation. It has been suggested that oxidized LDL phospholipids and cholesteryl esters are physiological substrates for serum paraoxonase [43]. One needs to wonder the need for such an esterase as several other enzymes, for example LCAT, PAF-acetyl hydrolase, and others could perform similar functions. Additionally, it has been shown that paraoxonase is able to reduce hydrogen peroxide (H2O2), a major reactive species produced during oxidative stress [6]. Considering the universal ‘‘acceptance’’ of esterase activity as a correlative parameter with atherosclerosis, it is tempting to speculate a concerted mechanism for the action of PON1 on the reduction of lipid hydroperoxides that would involve a hydrolysis of the esterified lipid to free fatty acid hydroperoxide followed by its reduction. If so, by preventing the oxidative inactivation of the esterolytic activity of PON1, suitable antioxidants would enhance the enzyme’s action on the detoxification of esterified lipid hydroperoxides. Considering that fat-soluble antioxidant vitamin E showed only a partial protection, whereas the water-soluble antioxidant vitamin C afforded little or no protection, there is a need for testing other water-soluble antioxidants.
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Acknowledgements This work was supported by NIH grant HL-069038 and DK-56353. We are grateful to Dr. Srinivasa Reddy, UCLA, for his generous gift of the antibody to PON1.
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