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1 Aville online Journl of Chemicl nd Phrmceuticl Reserch, 2014, 6(8): Reserch Article ISSN : CODEN(USA) : JCPRC5 The influence of dietic nd non-dietic very low density lipoproteins (VLDL) prticles on the modifiction of Low density lipoproteins (LDL) prticles Rveenn Mingpknee 1 nd Wini Dhln 2 1 Deprtment of Trnsfusion Medicine nd Microiology, Fculty of Allied Helth Sciences, Chullongkorn University, 154 Rm I Rod Pthumwn Bngkok, Thilnd 2 The Hll Science Center (HSC), Chullongkorn University, Fl CU Reserch Building 254 Phythi street Pthumwn Bngkok, Thilnd ABSTRACT Hypertriglyceridemi (HTG) is common compliction in type 2 dietes (DM) resulted from very low-density lipoprotein (VLDL) overproduction y the liver. Furthermore, VLDL prticles from HTG ptients crry more triglycerides (TG) thn VLDL prticles isolted from norml triglyceride level individuls, leding to n ccelerting trnsfer of TG from VLDL core to low-density lipoprotein (LDL) prticles. Triglyceride-rich LDL undergoes TG core hydrolysis y lipoprotein lipse nd ecome to smller nd denser prticles clled smll, dense LDL (sd-ldl). These prticles re more therogenic thn lrge, uoynt LDL (l-ldl). This study ws set up to study the influence of VLDL isolted from different hypertriglyceridemic sttus of dietic nd non-dietic donors. VLDL prticles were collected from dietic nd non-dietic donors which ctegorized y plsm TG; i.e., norml triglyceridemi (n-tg, plsm TG<150 mg/dl), high-triglyceridemi (h-tg, plsm TG etween mg/dl) nd very high-triglyceridemi (vh-tg, plsm TG>300 mg/dl). LDLs were incuted with different VLDL prticles in physiologicl condition. After 24-h incution, modified LDL (m-ldl) ecme TG-rich nd totl cholesterol (TC) poor prticles. The lipid profiles which found in modified LDL were similr to LDL isolted from HTG donors. Both VLDL isolted from HTG donors with dietic nd non-dietic ltered LDL lipid compositions in similr mnner, wheres LDL isolted from dietic donors with n-tg yielded similr therogenic modified LDL. This study demonstrted tht LDLs were le to e more therogenic in oth HTG nd type 2dietic sttes, despite the ltter eing in norml TG levels. Key words: type 2 dietes; hypertriglyceridemi; smll, dense LDL INTRODUCTION There is growing evidence from epidemiologic, clinicl nd lortory dt tht elevted plsm triglyceride (TG) levels re independent risk fctor for crdiovsculr disese [1,2,3,4]. However, hypertriglyceridemi (HTG) is ssocited with numer of lipid metolic disturnces, including predominnce in smll, dense low-density lipoprotein (sd-ldl) prticle [5,6]. Furthermore, HTG is common compliction in type 2 dietes mellitus [7,8]. The dyslipidemi pttern frequently oserved in type 2 dietes is incresed plsm TG levels, decresed highdensity lipoprotein (HDL) cholesterol levels, nd predominnce of sd-ldl [9,10].The mechnisms for the production of sd-ldl in dietic ptients re similr to those found in people with hypertriglyceridemi: increse in heptic lipse. Austin et l. hve shown tht low-density lipoproteins (LDL) suclss pttern B, which is chrcterized y prepondernce of sd-ldl prticles, is ssocited with n increse risk of myocrdil infrction nd tht it is correlted with incresed concentrtions of plsm TG nd low concentrtions of HDL cholesterol [11]. 545

2 Rveenn Mingpknee nd Wini Dhln J. Chem. Phrm. Res., 2014, 6(8): Hypertriglyceridemi is resulted from the overproduction of very low-density lipoproteins (VLDL) y the liver [12,13], leding to n ccelerted trnsfer of TG exchnges from VLDLs to low-density lipoproteins (LDL) nd vice vers cholesteryl ester (CE) from LDL to VLDL[14]. The TG-rich LDL prticles yielded from the exchnge undergo hydrolysis of TG core y heptic lipse which would finlly result in lipid-poor, protein-rich prticles. These LDLs were smller nd denser thn norml [15]. The differences of VLDL compositions isolted from norml nd HTG were identified. VLDL prticles isolted from HTG ptients contin higher TG thn VLDL prticles isolted from norml sujects. Furthermore, VLDL prticles from dietic ptients re lso TG-rich prticles. These VLDL prticles ccelerte core lipid trnsfer etween VLDL nd LDL, leding to generte more sd-ldl prticle [16].This experiments im to study the influence of VLDL isolted from different HTG sttes of dietic nd non dietic donors on the formtion of sd- LDL, in vitro. Two experiments were set up in order to investigte the ltertion of TG nd TC contents in modified LDL prticles. EXPERIMENTAL SECTION SUBJECTS Plsm lipids nd lipoproteins were collected from donors s the following criteri Group I: Non dietic sujects (n=15) (plsm glucose < 100 mg/dl) ctegorized y plsm TG level into 3 groups;1) Normotriglyceridemic (n-tg) group; plsm TG < 150 mg/dl (n=5), 2) high-triglyceridemic (h-tg) group; plsm TG etween mg/dl (n=5), nd 3)very high-triglyceridemic (vh-tg) group; plsm TG> 300 mg/dl (n=5). Group II: Type 2 dietic sujects (n=15) (plsm glucose > 130 mg/dl)ctegorized y plsm TG levelinto 3 groups; 1) Normotriglyceridemic (n-tg) group; plsm TG < 150 mg/dl (n=5), 2) high-triglyceridemic (h-tg) group; plsm TG etween mg/ dl (n=5), nd 3)very high-triglyceridemic (vh-tg) group; plsm TG > 300 mg/dl (n=5). The dietes ptients displyed vrying degree of glycemic control with HA1c rnging from 6.4 to 9.9% (reference %). A non-dietic helthy suject (totl cholesterol < 200 mg/dl, triglyceride < 150 mg/dl, LDL-C < 130 mg/dl, HDL > 50 mg/dl nd plsm glucose < 100 mg/dl) which not includes in ll study sujects ws recruited. LDL nd lipoprotein-poor plsm (contined lipid trnsfer proteins) were isolted from helthy suject used in the LDL modifiction experiments. Recruitment of sujects ws sed on extensive pulicity nd dvertisements t the Helth Sciences Service Unit, Chullongkorn University. This work ws pproved y the Ethicl Committee of Fculty of Medicine, Chullongkorn University. Written informed consent ws otined fter explntion of the purpose, nture nd potentil risks of this study to the sujects. The study ws conducted ccording to the requirements of the Ethicl Committee of Fculty of Medicine, Chullongkorn University. Blood smples were collected from ll donors in the morning fter 12-hour overnight fst y using EDTA (1 mg/ml lood) s nticogulnt. Plsm were isolted y centrifugtion t 3000xg 4 o C for 10 min. Lipoprotein nd lipoprotein poor plsm preprtions Lipoproteins were isolted from plsm y sequentil ultrcentrifugtion [17]. VLDL nd LDL were isolted t density rnge g/ml nd g/ml, respectively. Ultrcentrifugtion ws performed in himc CS100 Microultrcentrifuge (Hitchi KoKi, Tokyo, Jpn). LDL ws seprted into 2 su-frctions i.e. lrge, uoynt LDL (l-ldl) nd smll, dense LDL (sd-ldl) t density rnge g/ml nd g/ml, respectively. Lipoprotein-poor plsm ws seprted y sequentil ultrcentrifugtion t the density > 1.21 g/ml. This frction contins plsm proteins including lipid trnsfer proteins. VLDL nd LDL were deslted with PBS (ph 7.4) y gel filtrtion in Sephdex G-25M PD-10 columns (Amershm, USA). The protein content of the purified lipoprotein frction ws mesured y Lowry method [18], using ovine serum lumin s stndrd. Totl cholesterol nd triglyceride contents in unmodified VLDL nd modified VLDL, were determined y enzymtic methods (Humn, Germny) with UV-Visile spectrophotometer Evolution 300 (Thermo Scientific, USA). 546

3 Rveenn Mingpknee nd Wini Dhln J. Chem. Phrm. Res., 2014, 6(8): Experiment 1 Lipoprotein modifictions in vitro The VLDL nd LDL were modified, in vitro, y co-incution with lipoprotein-poor plsm in physiologicl concentrtion. VLDL (1 mg of proteins) were mixed with norml LDLisolted from helthy suject which not includes in ll study sujects(2 mg of proteins) nd lipoprotein-poor plsm (60 mg of proteins/ml) in totl volume of 5 ml contining 5 mm PBS uffer/ 1 mm EDTA, ph 7.4. The mixtures were incuted under N2 in slowly shking wter th t 37oC for 24 hrs. Allowed the lipid core exchnges etween VLDL nd LDL prticles. After incution, re-seprted lipoproteins y sequentil ultrcentrifugtion s previously descried. Isolte modified- VLDL (m-vldl) t density g/ml, wheres seprted the modified-ldl (m-ldl) t density rnge g/ml. Experiment 2 Lipoprotein lipse hydrolysis Modified-LDL enriched with TGws further core TG hydrolysis y purified ovine milk lipoprotein lipse (Sigm) in vitro. M-LDLs (4 mg proteins), 6% ovine serum lumin (ftty cid-free lumin), 5 mm PBS, ph 7.4 nd 10µL purified ovine milk lipoprotein lipse. The lipoprotein lipse ws immeditely dded fter mixing previous components. Incuted the mixture in shking wter th t 37oC for 1hr.After incution, the post hydrolytic modified-ldl (mp-ldl) were isolted y sequentil ultrcentrifugtion t density rnge g/ml. Totl cholesterol (TC) nd triglyceride contents in VLDL, m-vldl, unmodified LDL modified-ldl nd mp-ldl y using commercilly ville kits (Humn, Germny). LDL prticle dimeter determintions Unmodified LDL, modified-ldl nd mp-ldl prticle size were determined y electron microgrph (JEOL electron microscope model JEM 1230, USA) of negtive stin. Exmined smples t concentrtion of mg of protein/ml. Grids used in the experiment were prepred y the Scientific nd Technology Reserch Equipment Center, Chullongkorn University. Mesured the dimeters of prticles for size determintion nd the mens±se were clculted. RESULTS AND DISCUSSION Lipoprotein profile of study sujects The concentrtion of plsm lipids nd lipoproteins of non-dietes nd dietes donors were shown in Tles 1. It is shown tht oth non-dietic nd dietic donors with vh-tg hve the lowest HDL-C nd the highest sd-ldl-c levels, compred with other groups. Tle 1 Lipoprotein profiles of type 2 dietes nd non-dietes Type 2 Dietes Non dietes Lipoprotein n-tg h-tg vh-tg n-tg h-tg vh-tg (n=5) (n=5) (n=5) (n=5) (n=5) (n=5) TG (mg/dl) VLDL-TG LDL-TG l-ldl-tg sd-ldl-tg HDL-TG 72.1± ± ± ± ± ± ± ± ± ± ± ± ±33.9 c 379.7±25.2 c 50.5±4.7 c 30.9± ±1.9 c 35.3±7.0 c 103.9± ± ± ± ± ± ± ± ± ± ± ± ±20.0 c 447.3±33.8 c 71.9±9.2 c 39.7± ±2.5 c 55.4±9.7 CHL (mg/dl) VLDL-C LDL-C l-ldl-c sd-ldl-c HDL-C 200.9± ± ±9.0 86± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±6.8, 300.7± ± ± ± ± ±4.4 c Results re mens±se. TG=Triglyceride; CHL=Choleste rol; VLDL=Very low-density lipoproteins; LDL=low-density lipoproteins; l- LDL=lrge, uoynt low-density lipoprotein; sd-ldl=smll, dense low-density lipoprotein; HDL=high-density lipoproteins. Mens with different letters re significntly different from ech other t p<0.05. Modifiction of LDL y dietic nd non-dietic VLDL The effect of dietic nd non-dietic VLDL on LDL modifiction ws studied y the following experiments. VLDL ws isolted from n-tg, h-tg nd vh-tg donors with nd without dietic sttus. Figure1A nd 1B illustrted respective the TG nd TC contents in modified-ldl. After 24-h incution, the modified-ldl ws enriched in TG nd reduced in TC compred with the unmodified-ldl. The influences of VLDL isolted from nondietic nd dietic donors on LDL modifiction were compred. Normotriglyceridemic VLDL isolted from dietic nd non-dietic sujects yielded different LDL modifictions. Dietic n-tgvldl yielded higher TG content in modified-ldl compred with modified LDL induced y non-dietic n-tgvldl s shown in Figure 1A (4.4±0.2 vs 3.6±0.2 mg/mg proteins for LDL-TG induced y dietic nd non-dietic n-tgvldl, respectively, p<0.05). 547

4 Rveenn Mingpknee nd Wini Dhln J. Chem. Phrm. Res., 2014, 6(8): The TC contents in modified-ldl were shown in Figure 1B. Drmtic chnges were found in modified-ldl y TC decrese proportionlly to TG increse. So, LDL modified y vh-tg VLDL possessed the highest levels of TG with more loss of TC. Almost 50% of TC left in LDL fter eing modified y vh-tgvldl, wheres LDL modified y h-tgvldl nd n-tgvldl contin respective 63% nd 70% of TC compred with unmodified LDL. Non dietic nd dietic VLDL induced indifferent chnges in TC contents of modified LDL. 8 A Non dietic Dietic TG concentrtion in m-ldls (mg/mg proteins) p<0.05 p< unmodified Ntive LDL LDL LDL+ mldl nvldl with LDL+mHTG mldlwith VLDLDL+HTG mldl VLDL with n-tgvldl h-tgvldl vh-tg VLDL 14 B 12 Non dietic Dietic TC concentrtion in m-ldls (mg/mg proteins) c 2 0 unmodified Ntive LDL LDL LDL+ mldl nvldl with LDL+mHTG mldlwith VLDLDL+HTG mldl with VLDL Figure 1Comprison etween TG (A) nd TC (B) concentrtions in modified LDL (m-ldl) induced y non-dietic nd dietic VLDLs. The unmodified LDLs were modified y co-incution with non-dietic nd dietic VLDLs ctegorized y plsm TG: normotriglyceridemi(n-tg) VLDL, high-triglyceridemic (h-tg) VLDL nd very high-triglyceridemic (vh-tg) VLDL Ech r represents mens±se,,, = significn t difference within non-dietic group,,,c = significnt difference within dietic group. 548

5 Rveenn Mingpknee nd Wini Dhln J. Chem. Phrm. Res., 2014, 6(8): Incution of LDL with VLDL in the presence of lipoprotein poor plsm resulted in lipid composition chnges in LDL. Interestingly, the unmodified LDLs were modified y VLDLs isolted from normolipidemic donors in physiologicl condition. TG-rich lipoproteins served not only s donor for TGs to LDLs, ut lso s n cceptor for CE leving from the LDL core (Figure 1A nd 1B). The otined TG-enriched LDLs hd een modified upon lipolysis vi LpL, resulting in net loss of TGs nd generting smller LDL prticles. A similr study ws performed y severl groups [16,19]. A 8 Non dietic Dietic TG concentrtion in mp-ldls (mg/mg proteins) p<0.05 c 0 unmodified Ntive LDL LDL LDL+ mldl nvldl with LDL+mHTG mldlwith VLDLDL+HTG mldl VLDL with B 14 TC concentrtion in mh-ldls (mg/mg proteins) Non dietic Dietic 2 0 Ntive unmodified LDL LDL LDL+ nvldl mldl LDL+mHTG with mldlwit VLDLDL+HTG h VLDL mldl with Figure 2TG (A) nd TC (B) concentrtions in post-hydrolytic modified (mp) LDL. The modified-ldls were further TG core hydrolysis, in vitro, y lipoprotein lipse. Ech r represents mens±se,, = significnt difference within non-dietic group,,,c = significnt difference within dietic group Deckelum nd collegues studied the lipid trnsfer etween unmodified LDL or in vitro produced LDL-like prticles nd VLDLs. After incution, oth LDLs nd LDL-like prticles showed similr chnges. Furthermore, they incuted VLDLs with IDLs nd found tht the IDLs were modified y VLDLs similrly to LDLs. They 549

6 Rveenn Mingpknee nd Wini Dhln J. Chem. Phrm. Res., 2014, 6(8): suggested tht core lipid exchnge my occur etween different lipoproteins of vrying compositions nd physicl properties [16]. Furthermore, lipid trnsfer process occurred independently of polipoproteins present in oth lipoprotein prticles [20]. Chit nd his collegues found tht the modified LDL compositions were similr to compositions oserved in non-dietic nd dietic hypertriglyceridemic sttes [21]. In conclusion, the mount of TG trnsferred to LDL is depend on TG content in VLDL, furthermore, VLDL isolted from type 2 dietic ptients with norml triglycerides cn induce more triglyceride trnsfer to modified LDL. Triglyceride core hydrolysis of modified-ldls The modified LDLs derived from previous study were further TG core hydrolysis. Ifhigh TG core content were removed y hydrolysis, ecome post-hydrolytic modified LDL (mp-ldl), the mp-ldl ws smller nd denser prticle. Figure 2A represents TG contents in post-hydrolytic modified LDLs (mp-ldls). LDL whichmodified y VLDL isolted from non-dietic nd dietic donors wsfurther investigted for in vitrocore TG hydrolysis y lipoprotein lipse. It ws found tht the TG contents inmp-ldl fter TG hydrolysis ltered in different mgnitude compred with those without TG hydrolysis. The mp-ldl induced y dietic n-tgvldl mintined higher TG contents compred with those induced y non-dietic n-tgvldl (2.9±0.2 vs 2.3±0.1 mg/mg proteins, p<0.05) wheres, mp-ldls incuted with oth h-tgvldl nd vh-tgvldl of non-dietic nd dietic show reltively similr TG contents (3.5±0.1 vs 3.4±0.1 nd 5.0±0.1 vs 4.7±0.2 mg/mg proteins, for h-tgvldl nd vh-tgvldl, respectively). The TC concentrtions of mp-ldls were illustrted in Figure 2B. TC of mp-ldls remins constnt compred with modified-ldls prior to TG hydrolysis. LDL prticle dimeters The mp-ldls derived from previous study were further TG core hydrolysis. If high TG core content were removed y hydrolysis, ecome mp-ldl, the mp-ldl ws smller nd denser prticle. The dimeters of unmodified-ldl, m-ldl nd mp-ldl were mesured from prticles nd the mens±se were clculted. It is shown tht modified-ldl prticles enriched with TG hd wider dimeters. After TG hydrolysis, modified-ldls were then ltered to e post hydrolysis modified-ldls with nrrower dimeters (Figure 3). VLDL prticles re heterogeneous with regrd to its compositions: most of it is represented y lrge, TG-enriched or VLDL1 prticles, ut considerle frction is relesed s smller comprtively CE-enriched or VLDL2 prticles [22]. Normotriglyceridemic sujects contin reltively more VLDL2, wheres hypertriglyceridemic sujects were predominnt with VLDL1.23,24Due to the rte of VLDL1 secretion is dependent on TG vilility [25,26], hypertriglyceridemic sujects which hve over supply of TGs in the liver were, then, increse the VLDL1 overproduction. VLDL2 secretion my e more dependent on cholesterolsynthesis nd CE vilility [27]. To identify whether VLDLs isolted from hypertriglyceridemic lter LDL composition in vitro, VLDLs isolted from normo- nd hypertriglyceridemic donors with nd without dietic sttus were studied. Hypertriglyceridemic donors were ctegorized y fsting plsm TG; i.e., n-tg (TG<150 mg/dl), h-tg (TG mg/dl), nd vh-tg (TG>300 mg/dl). It ws found tht the degree of TG enrichment in m-ldl concomitnt with CE depletion vried with reltive to VLDL-TG concentrtions. As the VLDL-TG increse, the core lipid exchnges increses too, resulting in ccumultion of TG in m-ldls (Figure 1A). The TG nd TC contents of m-ldls induced y VLDLs isolted from hypertriglyceridemic donors in vitro were in similr pttern compred with unmodified LDLs isolted from hypertriglyceridemic donors [28,29]. It suggested tht TG trnsfer ws enhnced in hypertriglyceridemic sttes which produce TG-enriched LDL prticles. From this experiment, we found tht VLDLs isolted from dietic nd non-dietic hypertriglyceridemic donors were induced similr m-ldl compositions. Unexpectedly, VLDLs isolted from dietic normotriglyceridemic donors induced m-ldls prticles which more therogenic thn non-dietic normotriglyceridemic donors. LDLs modified y VLDLs isolted from dietic normotriglyceridemic donors contin high TG levels compred with those from non-dietic donors. This difference cn explin y VLDL heterogeneity. As previously descried, VLDLs re heterogeneous (VLDL1; TG-rich prticles nd VLDL2; CE-rich prticles).plsm VLDLs re regulted y insulin, which reduces the VLDL1 production in insulin sensitive individuls, ut hs minor effect on VLDL2. Insulin resistnce sujects or type 2 dietes re unle to inhiit the relese of VLDL1 from liver [30,31,32]. Although the VLDL su-frctions unidentified. It my suggest tht type 2 dietic donors contin more VLDL1 prticles nd induced more therogenic TG-rich LDLs. Further investigtion in normotriglyceridemic dietic donors is thus suggested in order to identify chrcteristics of their VLDL prticles in terms of protein nd lipid profiles using gs chromtogrph-mss spectrometry-mss spectrometry (GC-MS-MS) nd liquid chromtogrphmss spectrometry-mss spectrometry (LC-MS-MS) s tools. 550

7 Rveenn Mingpknee nd Wini Dhln J. Chem. Phrm. Res., 2014, 6(8): A 28 Non dietic Dietic Modified-LDL prticle dimeters (nm) c B 18 unmodified LDL mldl with mldlwithm LDL with Ntive LDL LDL+nTG-VLDLs LDL+mHTG-VLDLs LDL+sHTG-VLDLs 28 Mp-LDL prticles dimeter (nm) Non dietic Dietic 18 unmodified LDL mldl with mldlwith mldl with Ntive LDL LDL+nTG-VLDLs LDL+mHTG-VLDLs LDL+sHTG-VLDLs Figure 3 The dimeter (nm) of unmodified-ldl, modified-ldl nd post hydrolytic modified-ldl (mp-ldl) Ech r represents mens±se.,,, = significn t difference within non-dietic group,,,c = significnt difference within dietic group CONCLUSION Hypertriglyceridemi in presence nd sence dietic sttuses re induced similr chnge in modified-ldl TG concentrtions. These prticles undergo TG hydrolysis in vitro nd ecome smller. In contrst, normotriglyceridemic dietic sttus exggertes dyslipidemic sttus, even though dietic ptients hve norml plsm TG. From this experiment, we found tht VLDL isolted from dietic nd non-dietic hypertriglyceridemic donors were induced similr modified-ldl compositions. Unexpectedly, VLDLs isolted from dietic normotriglyceridemic donors induced modified-ldls prticles which more therogenic thn nondietic normotriglyceridemic donors. LDLs modified y VLDLs isolted from dietic normotriglyceridemic donors contin high TG levels compred with those from non-dietic donors. It suggests tht some difference etween non-dietic nd dietic VLDL prticles my induce different modified-ldl compositions. 551

8 Rveenn Mingpknee nd Wini Dhln J. Chem. Phrm. Res., 2014, 6(8): Acknowledgements This Reserch is supported y The Hll Science Center (HSC) Chullongkorn University, Thilnd Reserch Fund (TRF) nd Office of the Higher Eduction Commission under contct numer MRG REFERENCES [1] MA Austin, JE Hoknson, KL Edwrds.Am.J.Crdiol., 1998, 81, 7B-12B. [2] SM Grundy.Am. J.Crdiol 1998, 81, 18B-25B. [3] MA Austin, B McKnight, KL Edwrds, CM Brdley, MJ McNeely, BM Psty, et l. Circultion, 2000, 10, [4] G Yun, KZ Al-Shli, RA Hegele. Cn. Med. Assoc. J., 2007, 176, [5] MJ Stmpfer, RM Kruss, J M, PJ Blnche, LG Holl, FM Scks, CH Hennekens. JAMA., 1996, 276, [6] ZT Bloomgrden. Dietes Cre,2004, 27, [7] R Crmen. Am. Hert. J., 2005, 150, [8] WT Ceflu. Insulin Experimentl Biology nd Medicine, 2001, 226, [9] SM Hffner. Dietes Cre, 1998, 21, 160. [10] Hffner SM. Am. J.Crdiol., 1999, 83, 17F-21F. [11] MA Austin, JL Breslow, CH Hennekens, JE Buring, WC Willett, RM Kruss. JAMA, 1988, 260, [12] S Venktesn, P Cullen, P Pcy, D Hllidy, J Scott. Arterioscler. Throm. Vsc. Biol., 1993, 13, [13] C Tghiiglou, A Crpentier, SC Vn Iderstine, B Chen, D Rudy, A Aiton, et l. J. Biol.Chem.,2000, 275, [14] KK Berneis, RM Kruss. J. Lipid Res., 2002, 43, [15] RM Kruss. Am. J.Crdiol., 1998, 81, 13B-17B. [16] RJ Deckelum, S Eisenerg, Y Oschry, E Butul, I Shron, T Olivecron. J. Biol. Chem., 1982, 257, [17] RJ Hvel, HA Eder, JH Brgdon. J.Clin. Invest., 1955, 34, [18] OH Lowry, NJ Roserough, FA Lewis nd RJ Rndll.J. Biol. Chem.,1951, 193, [19] SH Qurfordt, F Boston, H Hildermn. Biochim. Biophys. Act, 1971, 231, [20] E Grnot, RJ Deckelum, S Eisenerg, Y Oschry, G Bengtsson-Olivecron. Biochim. Biophys. Act,1985, 833, [21] A Chit, S Eisenerg, A Steinmetz, JJ Alers, EL Biermn. Biochim. Biophys. Act, 1984, 795, [22] KG Prhofer, HR Brrett.J. Lipid Res., 2006, 47, [23] T St, RJ Hvel, AL Jones. J. Lipid Res., 1972, 13, [24] CJ Pckrd, J Shepherd, S Joerns, AJr Gotto, OD Tunton. Biochim. Biophys. Act, 1979, 572, [25] C Dchet, E Cvllero, C Mrtin, G Girrdot, B Jcotot. Atherosclerosis, 1995, 113,1-9. [26] CE Tn, L Forster, MJ Cslke, DT Bedford, DG Wtson, M McConnell, et.l. Arterioscler. Throm. Vsc. Biol.1995, 15, [27] RJ Pese, JM Leiper. Curr. Opin. Lipidol., 1996, 7, [28] NF Gleno, M Al-Hideri, F Keysermn, SC Rumsey, RJ Deckelum. J. Lipid Res., 1998, 39, [29] B Lmrche, S Rshid, GF Lewis. ClinChimAct, 1999,286, [30] R Mlmström, CJ Pckrd, TDG Wtson, S Rnnikko, M Cslke, D Bedford, et l. Arterioscl. Throm. Vsc. Biol., 1997, 17, [31] R Mlmström, CJ Pckrd, M Cslke, D Bedford, P Stewrt, H Yki-Järvinen, et.l. Dietologic,1997, 40, [32] S Bioletto, A Goly, R Munger, B Klix, RW Jmes. Am. J.Clin. Nutr., 2000, 71,

9 Rveenn Mingpknee nd Wini Dhln J. Chem. Phrm. Res., 2014, 6(8):