Anette Pedersen, 1, * Manfred W. Baumstark, Peter Marckmann,* Helena Gylling, and Brittmarie Sandström*

Transcription

1 An olive oil-rih diet results in higher onentrtions of LDL holesterol nd higher numer of LDL sufrtion prtiles thn rpeseed oil nd sunflower oil diets Anette Pedersen, 1, * Mnfred W. Bumstrk, Peter Mrkmnn,* Helen Gylling, nd Brittmrie Sndström* Reserh Deprtment of Humn Nutrition,* Center for Advned Food Studies, Royl Veterinry nd Agriulturl University, DK-1958 Frederikserg, Denmrk; Center for Internl Mediine, University of Freiurg, D Freiurg, Germny; nd Division of Internl Mediine, Deprtment of Mediine, University of Helsinki, Helsinki, Finlnd Astrt We investigted the effet of olive oil, rpeseed oil, nd sunflower oil on lood lipids nd lipoproteins inluding numer nd lipid omposition of lipoprotein sulsses. Eighteen young, helthy men prtiipted in doulelinded rndomized ross-over study (3-week intervention period) with 50 g of oil per 10 MJ inorported into onstnt diet. Plsm holesterol, triylglyerol, polipoprotein B, nd very low density lipoprotein (VLDL), intermedite density lipoprotein (IDL), nd low density lipoprotein (LDL) holesterol onentrtions were 10 20% higher fter onsumption of the olive oil diet ompred with the rpeseed oil nd sunflower oil diets [nlysis of vrine (ANOVA), P 0.05]. The size of IDL, VLDL, nd LDL sufrtions did not differ etween the diets, wheres signifintly higher numer (polipoprotein B onentrtion) nd lipid ontent of the lrger nd medium-sized LDL sufrtions were oserved fter the olive oil diet ompred with the rpeseed oil nd sunflower oil diets (ANOVA, P 0.05). Totl HDL holesterol onentrtion did not differ signifintly, ut HDL 2 holesterol ws higher fter olive oil nd rpeseed oil ompred with sunflower oil (ANOVA, P 0.05). In onlusion, rpeseed oil nd sunflower oil hd more fvorle effets on lood lipids nd plsm polipoproteins s well s on the numer nd lipid ontent of LDL sufrtions ompred with olive oil. Some of the differenes my e ttriuted to differenes in the squlene nd phytosterol ontents of the oils. Pedersen, A., M. W. Bumstrk, P. Mrkmnn, H. Gylling, nd B. Sndström. An olive oil-rih diet results in higher onentrtions of LDL holesterol nd higher numer of LDL sufrtion prtiles thn rpeseed oil nd sunflower oil diets. J. Lipid Res : Supplementry key words lood lipids holesterol dietry ftty ids dietry oils HDL holesterol insulin ishemi hert disese nonesterified ftty ids phytosterols squlene triylglyerols Dietry ftty id omposition influenes plsm lipids nd lipoproteins ssoited with the development of theroslerosis nd ishemi hert disese (IHD). The effet of dietry ftty ids on plsm totl holesterol, nd on low density lipoprotein (LDL) nd high density lipoprotein (HDL) holesterol, hs een sujet to mny studies nd predition lgorithms hve een developed (1 6). However, the onventionl risk mrkers of IHD re not suffiient for identifying individuls t high risk of IHD (7) nd dditionl lood prmeters suh s elevted fsting plsm insulin, polipoprotein B (pob), triylglyerols (TAG), nd nonesterified ftty ids (NEFA) hve een studied in reltion to n inresed risk of IHD (8 11). Very low density lipoprotein (VLDL) or/nd intermedite density lipoprotein (IDL) mss onentrtions hve een found to e relted to disese progression (12 15). Results from linil studies hve suggested strong reltionship etween predominne of smll, dense LDL prtiles in plsm nd n inresed risk of myordil infrtion or oronry theroslerosis s ssessed from ngiogrphy (9, 16 19), nd prospetive studies hve onfirmed this ssoition (20, 21). The inresed therogeniity of smll, dense LDL my e explined y lower inding ffinity to the LDL reeptor (22), inresed suseptiility to oxidtion (23), or n inresed pity for Arevitions: ALP, therogeni lipoprotein phenotype; ANOVA, nlysis of vrine; po, polipoprotein; CRP, C-retive protein; FC, free holesterol; HDL, high density lipoprotein; HL, hepti lipse; IDL, intermedite density lipoprotein; IHD, ishemi hert disese; LDL, low density lipoprotein; MUFA, monounsturted ftty ids; OO, olive oil; NEFA, nonesterified ftty ids; PL, phospholipid; PUFA, polyunsturted ftty ids; RO, rpeseed oil; SFA, sturted ftty ids; SO, sunflower oil; TAG, triylglyerol; VLDL, very low density lipoprotein. 1 To whom orrespondene should e ddressed. Journl of Lipid Reserh Volume 41,

2 inding to the intiml proteoglyns (24). A predominne of smll, dense LDL prtiles is lso min hrteristi of the therogeni lipoprotein phenotype (ALP), whih is onsidered highly ssoited with IHD (25, 26). Only limited informtion exists onerning the effet of diet on VLDL, IDL, nd LDL nd HDL sulsses. In study of helthy men, the sujets with predominntly smller LDL prtiles (pttern B) exhiited signifintly greter redution in the mss of medium- nd smll-sized LDL sufrtions ompred with those with predominntly lrger LDL prtiles (pttern A) when hnging from high ft to low ft diet (27). This indites tht individuls with n ALP (pttern B) my enefit more from dietry modifitions thn individuls without n ALP (pttern A) (27). In the sme group of sujets (28), high sturted ft intke ws ssoited with n inresed mss of lrge holesterol-enrihed LDL prtiles. n 3 ftty id supplementtion to hypertriglyeridemi sujets hs resulted in n inresed numer of lrge, uoynt LDL (29) nd n inresed LDL prtile size (30) ompred with seline. Thus, the limited ville evidene suggests tht the mount nd type of dietry ft influene the LDL sufrtion profile, ut it is not ler whether ommon edile vegetle oils leding to vrying lood lipid levels lso differ with respet to lipoprotein sulss profiles. In the present study, we therefore ompred the effets of three experimentl diets rih in different vegetle oils (olive oil [OO], rpeseed oil [RO], nd sunflower oil [SO]) on lood lipids nd on the numer, size, nd omposition of LDL nd HDL sufrtions. It ws hypothesized tht RO, euse of its lower ontent of sturted ftty ids (SFA) ompred with OO nd its higher ontent of n n 3 ftty id, would e ssoited with the most fvorle lipoprotein sulss profile. Furthermore, we expeted tht SO, euse of its higher ontent of polyunsturted ftty ids (PUFA), would led to lower lood lipid onentrtions, ut higher, less fvorle totl nd LDL:HDL holesterol rtios ompred with the OO nd RO diets. The effets on lood ogultion ftor VII (31) nd the oxidtion resistne of VLDL nd LDL prtiles (N. S. Nielsen, P. Mrkmnn, nd C-E. Høy, unpulished oservtions) were lso studied, ut re presented seprtely. SUBJECTS AND METHODS Sujets Eighteen mle students were reruited for the study y lol dvertisement. The sujets were ged yers (men, 24 yers), weighed from 62 to 99 kg (men, 79 kg), nd hd ody mss indexes from 18 to 27 kg/m 2 (men, 23 kg/m 2 ). All sujets were nonsmokers nd did not use ny medition. They were pprently helthy, did not exerise exessively, nd hd no fmily history of therosleroti disese or hypertension. Men fsting lipid onentrtions t inlusion were s follows: plsm totl holesterol, 4.74 mm (rnge, 3.09 to 6.25 mm); HDLholesterol, 1.10 mm (rnge, 0.84 to 1.50 mm); nd plsm totl TAG, 1.2 mm (rnge, 0.41 to 3.29 mm). The im of the study ws explined orlly to eh sujet nd written informtion ws given, efore the sujets gve their written onsent. The reserh protool ws pproved y the Sientifi Ethis Committee of the muniiplities of Copenhgen nd Frederikserg (01-272/95). Experimentl design The study ws performed s doule-linded, rndomized, ross-over experiment with three periods of 3 weeks seprted y wsh-out periods of 5 12 weeks. Six groups of three sujets onsumed the diets in the following order: OO, RO, SO/OO, SO, RO/RO, OO, SO/RO, SO, OO/SO, OO, RO/SO, RO, OO, respetively. Sujets were refully instruted not to hnge hitul diets during the wsh-out periods nd to keep the sme physil tivity level during the study. Sujets reeived ll foods from our deprtment nd were not llowed to onsume other foods in the study periods, exept for wter, plin offee, nd te (offee nd te in smll mounts). Sujets were lloted to fixed energy levels ording to ody weight, ge, nd physil tivity indexes. Body weight ws mesured every seond dy in eh period, nd weight vrition of more thn 1 kg resulted in either the llotion to different energy level or to onsumption of extr rolls (sme nutrient omposition s the totl diet). The men energy intke during the study ws 15.4 MJ/dy (rnge, MJ). Consumption of extr rolls ws noted in diry, s were dily reords of physil tivity, ny sign of illness, medition, offee nd te intke, nd ny devition from the diet. On weekdys lunh ws onsumed in the deprtment under supervision, wheres everges, dinner, snk, nd rekfst for the next morning were provided dily s pkge with guidelines for its preprtion. Food nd everges for the weekend were provided on Fridys. Leftovers were rought k to the deprtment to e registered. The mounts of returned foods were smll nd did not ffet the verge ft intke nd ft omposition for ny of the sujets. Most food for eh intervention period ws prepred in one th in the metoli kithen, weighed, oded in olor ording to the oil used, nd frozen until use. Duplite portions of eh diet were olleted during 2 weeks in eh period of the study. The portions were pooled per diet nd nlyzed for energy ontent nd nutritionl omposition. Diet The three diets were identil exept for the qulity of ft. Nineteen perent of the totl dietry energy intke, tht is, 50 g/ 10 MJ, derived from either extr virgin OO (Nvrino, Dnton Trding, Århus, Denmrk), physilly refined RO (in pilot sle) (Deprtment of Biotehnology, Tehnil University of Denmrk, Lyngy, Denmrk), or hemilly refined SO (Solex W, Århus Olie, Århus, Denmrk). All other food items were held onstnt nd identil in the three experimentl diet periods. Menus were repeted every week nd onsisted of ommon prepred nd ooked foods. The test oils were inluded in red, rolls, kes, dressing, nd dinner dishes onsisting of vegetles, met, pst, rie, or pottoes. Food nlysis Duplite portions of eh of the three 3-week diets were olleted, homogenized, lyophilized, nd hemilly nlyzed. Nitrogen ws determined ording to the priniple of Dums (32) on n utomti nitrogen nlyzer (NA 1500; Crlo Er Strumentzione, Miln, Itly). Ft ontent ws mesured fter extrtion ording to the proedure y Folh, Lees, nd Stnley (33), nd the ontent of dietry fier ws determined enzymtilly (34). The diet mronutrient omposition is shown in Tle 1. The ftty id omposition of the 3-week diets ws nlyzed y gs hromtogrphy (8420; Perkin-Elmer, Birkerød, Denmrk) fter extrtion with diethyl ether-petroleum ether nd suse Journl of Lipid Reserh Volume 41, 2000

3 TABLE 1. Anlyzed mronutrient omposition (% of totl energy) nd fier nd holesterol ontent (g/10 MJ) of olive oil, rpeseed oil, nd sunflower oil diets OO Diet RO Diet SO Diet Ft Sturted ftty ids Monounsturted ftty ids Polyunsturted ftty ids Crohydrtes Protein Fier (g/10 MJ) Cholesterol (mg/10 MJ) Arevitions: OO, olive oil; RO, rpeseed oil; SO, sunflower oil. The holesterol ontent of the experimentl diet without dded oil ws lulted y the use of ntionl dtse (Dnkost 2.0, Dnish Ctering Center A/S, Herlev, Denmrk) nd the nlyzed holesterol ontent of the oils ws dded. quent methyltion with methnoli BF 3 (35) y the Ntionl Food Administrtion in Denmrk (Tle 2). The squlene nd sterol ontents of the oils (Tle 3) were determined from nonsponifile mteril y gs-liquid hromtogrphy on 50-mlong pillry olumn (Ultr 1 R ; Hewlett-Pkrd, Plo Alto, CA) s trimethylsilyl derivtives, using 5 -holestne s internl stndrd (36), t the University of Helsinki (Helsinki, Finlnd). Blood smpling Fsting lood smples were tken fter 10 min of supine rest efore the study nd on dys 21 nd 22 in eh diet period. Blood smples were olleted in tues without dditives for the nlysis of serum C-retive protein (CRP), nd in ethylenediminetetreti id tues for the nlysis of plsm insulin, NEFA, holesterol, TAG, polipoproteins, squlene, sterols, ftty id omposition of plsm holesteryl esters, nd lipoprotein frtiontion. All plsm smples were immeditely pled on ie, nd entrifuged t 3,000 g for 15 min t 4 C. Smples for serum CRP were kept t 20 C, smples for plsm insulin, NEFA, squlene, sterols, nd ftty id omposition of plsm holesterol esters t 80 C, nd smples for lipids, polipoproteins, nd lipoprotein frtiontion were shipped the sme dy (to Freiurg University, Freiurg, Germny) nd kept ooled (4 C) until nlysis. Elevted CRP onentrtion in one sujet on one osion nd tehnil prolems with smples from one sujet on one osion resulted in the exlusion of these oservtions. TABLE 2. Anlyzed ftty id omposition (mol%) of the three experimentl diets ontining 50 g/10 MJ of olive oil, rpeseed oil, nd sunflower oil Ftty Aids OO Diet RO Diet SO Diet Sturted ftty ids, totl Luri id (C 12:0 ) Myristi id (C 14:0 ) Plmiti id (C 16:0 ) Steri id (C 18:0 ) Monounsturted ftty ids, totl Olei id (C 18:1, n 9) Polyunsturted ftty ids, totl Linolei id (C 18:2, n 6) Linoleni id (C 18:3, n 3) Arevitions: OO, olive oil; RO, rpeseed oil; SO, sunflower oil. TABLE 3. Sterol nd squlene onentrtions of the test oils (mg/kg) Olive Oil Rpeseed Oil Sunflower Oil Cholesterol Cmpesterol 55 2, Sitosterol 958 3,892 2,417 Squlene 3, Blood nlysis Serum CRP ws ssessed y ommeril immuno-turidimetri method (Rohe, Bsel, Switzerlnd), plsm insulin y n enzymelinked immunosorent ssy method (Dko, Glostrup, Denmrk), nd plsm NEFA y ommeril enzymti olorimetri method (Wko Chemils GmH, Neuss, Germny). The ftty id omposition of plsm holesteryl esters ws determined fter seprtion y thin-lyer hromtogrphy. Plsm holesteryl esters were methylted with BF 3 (35) nd the methyl esters were nlyzed y gs hromtogrphy (HP 6890; Hewlett-Pkrd) t the Tehnil University of Denmrk. Plsm squlene nd nonholesterol sterols were determined s desried in the food nlysis setion. VLDL (d g/ml), IDL ( g/ml d g/ml), LDL (1.019 g/ml d g/ml), nd HDL (1.063 g/ml d 1.21 g/ml) were prepred y sequentil flottion ording to Lindgren et l. (37, 38). HDL sufrtions (HDL 2, g/ml d g/ml; HDL 2, g/ml d g/ml; HDL 3, g/ml d 1.21 g/ml) were prepred y equilirium density grdient entrifugtion ording to Anderson et l. (39, 40) with minor modifitions. Totl LDL ws frtionted into six density lsses y equilirium density grdient entrifugtion s desried previously (41). The density rnges of the sufrtions s determined y preision refrtometr y (38) of lnk grdients were s follows: LDL-1, d 1.031; LDL-2, d 1.034; LDL-3, d 1.037; LDL-4, d 1.040; LDL-5, d 1.044; LDL-6, d g/ml. All entrifugtion steps were performed t temperture of 18 C using prtilly filled (6 ml) polyronte ottles in 50 Ti rotor. Phospholipid (PL), free holesterol (FC), totl holesterol (TC), nd TAG were mesured y utomted (EPOS; Eppendorf, Hmurg, Germny) enzymti methods. Regent kits for TC nd TAG were otined from Boehringer Mnnheim (Mnnheim, Germny), kits for FC were purhsed from Wko Chemils, nd kits for PL were from iomérieux (Nürtingen, Germny). Apolipoprotein A-I (poa-i) nd polipoprotein A-II (poa-ii) were mesured y end-point nephelometr y (Behring, Mrurg, Germny). ApoB ws stndrdized ording to the Centers for Disese Control (CDC, Atlnt, GA) stndrd. This stndrd ws onfirmed y mino id nlysis in n LDL-3/ LDL-4 pool (41). Prtile omposition of pob-ontining prtiles ws ssessed y lulting the molr rtio of lipid per pob, using previously pulished moleulr weights (41). The verge prtile volume of eh sufrtion ws lulted from the ovedesried numer of lipid moleules per prtile nd the volume of the lipid moleules nd the estimted volume of one pob moleule (41). A rdius orresponding to this volume ws lulted, ssuming spheril lipoprotein prtile. Rdii lulted in this wy re in good greement with rdii mesured y X-ry smll-ngle sttering (41). The desried method for the nlysis of holesterol nd TAG in lipoproteins resulted in men reovery for holesterol of 0.85 (rnge, ) nd for TAG of 0.91 (rnge, ). The disrepny my e due to the ft tht onentrtions in plsm were mesured in smples other thn those used to determine onentrtions in lipoproteins. HDL holesterol ws lso mesured y lssi polyethylene glyol preipittion method (Quntolip; Immuno AG, Vienn, Austri) nd resulted in slightly higher HDL holesterol levels thn those presented in Pedersen et l. Dietry oils nd lipoprotein sufrtions 1903

4 this rtile. However, neither of the methods reveled signifint differenes in HDL holesterol etween the diets. In ddition, totl, hylomiron, VLDL, nd LDL plus HDL holesterol onentrtions were mesured in different lortor y (Reserh Deprtment of Humn Nutrition, Frederikserg, Denmrk) (fter seprtion y sequentil ultrentrifugtion) with kits from Boehringer Mnnheim nd the results showed lower holesterol onentrtions in totl plsm nd higher onentrtions in the individul lipoproteins, resulting in etter reover y for these dt. However, the sttistil nlyses of the two sets of dt resulted in similr results. Therefore, it ws deided tht the dt inluding mesurements of sufrtions should e presented despite risk of minor ompositionl is due to the inomplete reovery. The intr-ssy oeffiients of vrition for the determintions were s follows: insulin, 5.0%; NEFA, 1.8%; sterols, 2.5%; LDL sufrtion onentrtions of FC, PL, nd TC, 2 4.5%; of TAG, 4 8%; nd of pob, %, depending on the sufrtion. The interssy oeffiients of vrition were s follows: insulin, 5.9%; NEFA, 1.7%; sterols, 4.8%. Sttistil nlysis Results from eh diet intervention period were ompred y nlysis of vrine (ANOVA). If the ANOVA indited signifint differenes (P 0.05), it ws followed y post-ho sttistil nlysis using modified t-tests ording to the Bonferroni method (P vlues ove 0.1 were not Bonferroni orreted). Plsm TAG, HDL 2 holesterol, nd holesterol ester linolei id onentrtions were not distriuted normlly nd were log trnsformed. No period or rryover effets were oserved. Power lultions (power 0.85, signifine level P 0.05) showed tht with smple size of 18 sujets it should e possile to detet 0.2-mmol/l differene in totl plsm holesterol nd 0.1-mmol/l differene in plsm TAG (42). The SAS sttistil pkge (SAS Institute, Cry, NC) ws used for the sttistil nlyses. RESULTS Plsm onentrtions of lood lipids, polipoproteins, insulin, nd NEFA Fsting plsm totl holesterol onentrtions were pproximtely 12% higher fter onsumption of the OO TABLE 4. Blood lipids, lipoproteins, nd lipid rtios in 18 helthy men fter 3 weeks of onsuming diet rih in olive oil, rpeseed oil, or sunflower oil OO Diet RO Diet SO Diet mm Plsm totl triylglyerol 0.86 (0.07) 0.73 (0.04) 0.72 (0.05) Plsm totl holesterol 4.15 (0.18) 3.67 (0.19) 3.74 (0.16) VLDL holesterol 0.33 (0.03) 0.25 (0.02) d 0.27 (0.02) LDL holesterol 2.16 (0.14) 1.73 (0.14) e 1.89 (0.11) e HDL holesterol 0.97 (0.05) 0.98 (0.06) 0.90 (0.05) LDL:HDL holesterol 2.35 (0.22) 1.87 (0.19),f 2.23 (0.20) Totl:HDL holesterol 4.44 (0.32) 3.90 (0.28) 4.31 (0.28) Arevitions: OO, olive oil; RO, rpeseed oil; SO, sunflower oil; VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein. Sttistil nlysis: ANOVA followed y pirwise omprison y Student s t-test with Bonferroni orretion. Vlues re mens (SEM). P 0.05, d P 0.01, e P ompred with olive oil. f P 0.05 ompred with sunflower oil. diet ompred with the RO nd SO diets (ANOVA, P ), nd fsting plsm TAG nd pob onentrtions were pproximtely 20% higher fter OO ompred with RO nd SO (ANOVA, P 0.003); see Tle 4 nd Fig. 1. Plsm totl holesterol nd TAG nd pob onentrtions did not differ etween the RO nd SO diets. Plsm poa-i onentrtions were higher fter OO nd RO thn fter SO (ANOVA, P 0.002) (Fig. 1). Plsm poa-ii onentrtions did not differ signifintly etween the three diets [men (SEM), mg/dl; OO, 38.6 (1.1); RO, 36.5 (1.2); SO, 36.2 (1.0)], nor did fsting plsm insulin [men (SEM), pm; OO, 30.6 (1.9); RO, 29.3 (1.8); SO, 28.4 (1.2)]. Plsm NEFA onentrtions tended to e higher fter OO thn fter SO, while no signifint differene ws oserved etween RO nd the other oils [men (SEM), mm; OO, 0.40 (0.04); RO, 0.38 (0.04); SO, 0.33 (0.03); ANOVA, P 0.05; OO vs. SO, P 0.07]. VLDL, IDL, totl LDL The onentrtions of holesterol, TAG, PL, nd pob in VLDL, IDL, nd LDL re shown in Tle 5. The VLDL, IDL, nd LDL holesterol nd PL onentrtion were signifintly higher fter the OO diet thn fter the RO nd SO diets. The VLDL TAG onentrtion ws signifintly higher fter OO thn fter SO, nd the LDL TAG onentrtion ws signifintly higher fter OO thn fter RO nd SO (Tle 5). The onentrtion of pob in VLDL, IDL, nd totl LDL prtiles, orresponding to the numer of lipoprotein prtiles, ws signifintly higher fter the OO diet thn fter the RO nd SO diets, while no differene in pob onentrtion ws oserved etween the RO nd SO diets (Tle 5). The numer of lipid moleules in eh lipoprotein prtile is given in Tle 6. No signifint differene in the numer of holesterol moleules in eh VLDL or LDL prtile ws oserved. The numer of holesterol moleules (nd holesteryl ester, dt not shown) in eh IDL prtile ws higher fter OO thn fter RO (ANOVA, P 0.04; OO vs. RO, P 0.09), while the numer of TAG moleules in eh IDL prtile ws signifintly lower fter OO thn fter SO, with results fter RO intermedite. As onsequene of the virtully unhnged lipid omposition in the lipoprotein prtiles, the lulted verge size of VLDL, IDL, nd LDL did not differ etween the three diets [men rdius (SEM), nm: VLDL, 16.1 (0.2); IDL, 12.0 (0.1); LDL, 9.7 (0.1)]. LDL sufrtions The holesterol, TAG, nd PL onentrtions in the LDL sulsses exhiited the sme pttern through ll sulsses: onentrtions were higher fter OO thn fter RO nd SO, with results fter SO intermedite. With few exeptions the differenes were signifint for the lrger (LDL-1, LDL-2) nd medium-sized (LDL-3, LDL-4) LDL sufrtions nd not signifint for the smll, dense LDL prtiles (LDL-5, LDL-6). Cholesterol nd PL onentrtions were signifintly higher fter OO thn fter RO in LDL-1 to LDL-4, nd higher fter OO thn fter SO in 1904 Journl of Lipid Reserh Volume 41, 2000

5 Fig. 1. Individul nd men onentrtions of plsm holesterol, TAG, pob, nd poa-i in 18 helthy mles fter 3 weeks of onsuming OO, RO, or SO. LDL-1 to LDL-3. LDL-1, LDL-2, nd LDL-3 TAG onentrtions were higher fter OO thn fter RO nd SO. No signifint differenes in holesterol, TAG, nd PL etween the RO nd SO diets were oserved. The onentrtion of pob (orresponding to numer of LDL sufrtions) ws signifintly higher fter OO thn fter RO in LDL-1 to LDL-5, nd signifintly higher fter OO thn fter SO in LDL-1 to LDL-3 (Fig. 2). The pob onentrtion nd the lipid ontent of LDL sulsses did not differ etween the RO nd SO diets. The numer of holesterol moleules per LDL prtile did not differ signifintly etween the diets for ny LDL sufrtion (Tle 6). However, in ll LDL sulsses, the numer of TAG moleules per prtile ws higher fter RO thn fter OO. This ws sttistilly signifint for totl LDL, LDL-1, LDL-3, nd LDL-6. There were no differenes in the lulted verge size of the TABLE 5. Lipid nd polipoprotein B onentrtion in pob-ontining lipoproteins in 18 helthy mles fter 3 weeks of onsuming diet rih in olive oil, rpeseed oil, or sunflower oil Cholesterol Triylglyerols Phospholipids Apolipoprotein B OO RO SO OO RO SO OO RO SO OO RO SO VLDL 0.33 (0.03) IDL 0.12 (0.02) LDL 2.16 (0.14) mm mm mg/dl mg/dl 0.25 d (0.02) 0.08 d (0.01) 1.73 e (0.14) 0.27 (0.02) 0.08 d (0.01) 1.89 e (0.11) 0.56 (0.05) 0.04 (0.003) 0.10 (0.007) 0.46 (0.03) 0.04 (0.002) 0.09 (0.005) 0.47 (0.04) 0.04 (0.003) 0.09 d (0.004) 15.8 (1.4) 3.3 (0.4) 49.9 (3.0) 12.7 d (0.9) 2.5 d (0.2) 40.6 e (2.9) 13.0 (1.1) 2.6 (0.2) 43.2 e (2.4) 4.4 (0.4) 2.0 (0.3) 50.3 (3.3) 3.5 d (0.2) 1.5 (0.1) 39.5 e (2.7) 3.5 d (0.3) 1.4 d (0.1) 42.7 d (2.8) Arevitions: OO, olive oil; RO, rpeseed oil; SO, sunflower oil; VLDL, very low density lipoprotein; IDL, intermedite density lipoprotein; LDL, low density lipoprotein. Sttistil nlysis: ANOVA followed y pirwise omprison y Student s t-test with Bonferroni orretion. Vlues re mens (SEM). P 0.05, d P 0.01, e P ompred with olive oil. Pedersen et l. Dietry oils nd lipoprotein sufrtions 1905

6 TABLE 6. Numer of lipid moleules per prtile (molr rtio of lipid to pob) in 18 helthy mles fter 3 weeks of onsuming diet rih in olive oil, rpeseed oil, or sunflower oil Cholesterol Triylglyerols OO RO SO OO RO SO VLDL 3,840 (109) 3,687 (84) 3,924 (142) 6,739 (269) 6,766 (170) 6,754 (259) IDL 2,947 (103) 2,707 (96) 2,889 (93) 1,315 (83) 1,511 (75) 1,615 (97) d LDL 2,215 (32) 2,250 (45) 2,301 (57) 109 (5) 127 (6) d 114 (4) LDL-1 2,643 (50) 2,684 (73) 2,815 (60) 156 (10) 194 (12) 183 (12) LDL-2 2,469 (43) 2,587 (57) 2,565 (54) 104 (8) 120 (9) 107 (7) LDL-3 2,296 (49) 2,422 (48) 2,458 (60) 84 (6) 101 (7) 89 (4) LDL-4 2,241 (34) 2,359 (48) 2,330 (49) 85 (4) 97 (7) 87 (4) LDL-5 2,103 (32) 2,187 (40) 2,193 (46) 88 (4) 98 (6) 92 (4) LDL-6 1,821 (34) 1,855 (43) 1,813 (38) 126 (6) 144 (8) 131 (5) Arevitions: OO, olive oil; RO, rpeseed oil; SO, sunflower oil; VLDL, very low density lipoprotein; IDL, intermedite density lipoprotein; LDL, low density lipoprotein. Sttistil nlysis: ANOVA followed y pirwise omprison y Student s t-test with Bonferroni orretion. Vlues re mens (SEM). P 0.05, d P 0.01 ompred with olive oil. LDL sufrtions etween the three diets [men dimeter (SEM), nm: LDL-1, 10.3 (0.1); LDL-2, 10.0 (0.1); LDL-3, 9.8 (0.1); LDL-4, 9.7 (0.1); LDL-5, 9.6 (0.1); LDL- 6, 9.3 (0.1)]. HDL Totl HDL holesterol tended to e higher fter OO nd RO thn fter SO, lthough this differene ws not sttistilly signifint (ANOVA, P 0.06). The lipid (holesterol, TAG, nd PL) nd polipoprotein (poa-i nd poa-ii) ontents in HDL 2 were signifintly higher fter OO nd RO thn fter SO, wheres higher TAG nd PL (OO) onentrtions were oserved fter OO nd SO ompred with RO in HDL 3 (Tle 7). No signifint differenes in holesterol, TAG, nd PL onentrtion in HDL, HDL 2, or HDL 2 etween the OO nd RO diet were oserved. Fig. 2. Conentrtion of pob in LDL sufrtions in 18 helthy mles fter 3 weeks of onsuming OO (hthed), RO (ler), or SO (horizontl lines). Sttistil nlysis: ANOVA followed y pirwise omprison y Student s t-test with Bonferroni orretion. P 0.05, P 0.01, P ompred with OO. The higher poa-i onentrtion in plsm fter OO nd RO ompred with SO ws ompnied y inresed onentrtions of poa-i nd poa-ii in HDL 2 fter OO nd RO (Tle 8). No other differenes in poa-i or poa-ii in the HDL sufrtions etween the diets were oserved. Rtios The LDL holesterol:hdl holesterol rtio ws signifintly higher fter OO nd SO ompred with RO (Tle 4). Similrly, the totl holesterol:hdl holesterol rtio, referred to s n therogeni index (43), ws signifintly higher fter OO thn fter RO. The totl:hdl holesterol rtio ws higher fter SO ompred with RO, ut only with orderline signifine (ANOVA, P 0.01; RO vs. SO, P 0.06); see Tle 4. Plsm squlene nd sterol onentrtions The plsm squlene nd nonholesterol sterol onentrtions re presented s rtios to plsm holesterol (100 mmol/mol of holesterol) in order to eliminte the vrition in holesterol euse the sterols re trnsported minly in LDL. The plsm rtios of the holesterol preursors squlene nd desmosterol were signifintly higher fter the OO diet ompred with the RO nd SO diets (Tle 9). The plsm 8 -lthosterol:holesterol rtio ws higher fter OO nd RO ompred with SO, wheres the plsm 7 -lthosterol:holesterol rtio ws higher fter RO ompred with SO. The plsm sitosterol:holesterol rtio ws signifintly lower fter onsumption of the OO diet ompred with the RO nd SO diets, wheres the plsm mpesterol:holesterol rtio ws signifintly different etween ll three diets: highest fter the RO diet nd lowest fter the OO diet, with intermedite onentrtions fter the SO diet (Tle 9). Ftty id omposition of plsm holesterol esters The ftty id omposition of holesteryl esters in plsm ws nlyzed to ssess dietry ompline. The results onfirm high ompline. The olei, linolei, nd -linoleni id onentrtions in plsm holesteryl esters losely re Journl of Lipid Reserh Volume 41, 2000

7 TABLE 7. Lipid onentrtion in HDL frtions in 18 helthy mles fter 3 weeks of onsuming diet rih in olive oil, rpeseed oil, or sunflower oil Cholesterol Triylglyerols Phospholipids OO RO SO OO RO SO OO RO SO mm mm mg/dl HDL 0.97 (0.05) 0.98 (0.06) 0.90 (0.05) (0.003) (0.003) (0.002) 55.8 (2.8) 54.6 (3.0) 48.4 (2.9) HDL (0.03) 0.25 (0.03) 0.22 (0.03) (0.001) (0.001) (0.001) 12.1 (1.8) 12.7 (1.7) 10.6 (1.4) HDL (0.02) 0.33 (0.02) 0.29 (0.02) (0.001) (0.001) (0.001) 20.2 (1.2) d 20.1 (1.4) d 17.0 (1.3) HDL (0.01) 0.39 (0.01) 0.39 (0.01) (0.002) (0.001) (0.001) e 23.1 (0.6) 21.5 (0.6) 20.9 (0.8) Arevitions: OO, olive oil; RO, rpeseed oil; SO, sunflower oil; HDL, high density lipoprotein. Sttistil nlysis: ANOVA followed y pirwise omprison y Student s t-test with Bonferroni orretion. Vlues re mens (SEM). P 0.05, d P 0.01 ompred with sunflower oil. e P 0.05 ompred with rpeseed oil. fleted the presene of the prtiulr ftty id in the test diets (31). In ordne, the olei:linolei id rtio in plsm holesteryl esters ws highest fter onsumption of OO nd lowest fter onsumption of SO, with intermedite vlues fter onsumption of RO [men (SEM): OO, 0.62 (0.03); RO, 0.34 (0.002); SO, 0.20 (0.01) ANOVA, P ; ll omprisons, P ]. DISCUSSION In this stritly ontrolled dietry experiment in helthy men, we studied the effets of onsumption of diets rih in monounsturted ftty ids (MUFA) (OO nd RO) or in PUFA (SO) on numer, size, nd omposition of LDL nd HDL sulsses. In ddition, the effets on totl holesterol, VLDL, IDL, LDL, nd HDL holesterol, nd plsm insulin, TAG, nd NEFA were studied. For mny of the mesured vriles the effet of OO differed distintly from the effet of the other two oils. The sujets seline plsm holesterol onentrtions were reltively low (men, 4.74 mm), ut lose to those oserved for this ge group in lrge popultion studies in Denmrk (44). In ddition, previous studies hve shown tht hnges in dietry ftty id omposition ffet lood lipid onentrtions even in those sujets with low initil onentrtions (45, 46). In this study, nine (50%) of the sujets hd plsm holesterol 5 mm. However, their responses to the three experimentl diets were similr to those with plsm holesterol 5 mm. The OO diet resulted in higher onentrtions of VLDL, IDL, nd LDL prtiles (numer nd lipid ontent) thn the RO nd SO diets. The oserved differenes were sttistilly signifint for VLDL, IDL, totl LDL, nd the lrge nd medium-sized LDL sufrtions (LDL-1 to LDL-4). No signifint differenes etween the diets were oserved in numer or omposition of the smllest, dense LDL-6 prtiles, ut OO ompred with RO onsumption resulted in higher numer of the smll LDL-5 (Fig. 2). In ontrst to the oserved strong ssoition etween predominne of smll, dense LDL nd n inresed risk of IHD, it is urrently unknown to wht extent n inresed numer of lrge LDL prtiles, s oserved fter the OO diet, is unfvorle in regrd to IHD. A study of normolipidemis reported tht LDL from sujets with predominne of lrge or smll LDL prtiles exhiited weker LDL reeptor-inding ffinity thn LDL from sujets with predominne of medium-sized LDL prtiles (47). In ddition, inresed onentrtions of lrge, uoynt LDL prtiles hve een found in sugroups of ptients with IHD (48, 49). One study demonstrted n inresed LDL prtile size fter n 3 ftty id supplementtion (30). In this study, n RO rih diet ontining reltively high mount of -linoleni id did not ffet the size of the LDL sufrtions (or ny other lipoproteins) differently thn the OO TABLE 8. Apolipoprotein onentrtion in HDL frtions in 18 helthy mles fter 3 weeks of onsuming diet rih in olive oil, rpeseed oil, or sunflower oil Apolipoprotein A-I Apolipoprotein A-II OO RO SO OO RO SO mg/dl mg/dl HDL 91.4 (3.3) 90.4 (4.0) 84.3 (4.3) 30.7 (1.0) 28.6 (1.2) 28.5 (1.1) HDL (2.0) 12.5 (2.3) 9.8 (1.5) 2.8 (0.3) 2.8 (0.3) 2.8 (0.2) HDL (1.5) 26.0 (1.8) 23.1 (1.8) 9.4 (0.5) 9.6 (0.5) 8.5 (0.6) HDL (1.7) 49.8 (1.6) 50.3 (2.2) 20.5 (0.9) 19.2 (0.6) 20.2 (0.9) Arevitions: OO, olive oil; RO, rpeseed oil; SO, sunflower oil; HDL, high density lipoprotein. Sttistil nlysis: ANOVA followed y pirwise omprison y Student s t-test with Bonferroni orretion. Vlues re men (SEM). P 0.05 ompred with sunflower oil. Pedersen et l. Dietry oils nd lipoprotein sufrtions 1907

8 TABLE 9. Plsm squlene nd nonholesterol sterols in 18 helthy mles fter 3 weeks of onsuming diet rih in olive oil, rpeseed oil, or sunflower oil Olive Oil Rpeseed Oil Sunflower Oil mmol/mol of holesterol 10 2 Squlene 58 (3) 37 (2) 31 (2) 8 -Lthosterol 20 (2) 20 (1) 16 (1) d Desmosterol 98 (4) 70 (3) 73 (4) 7 -Lthosterol 169 (11) 181 (8) 156 (8) e Cholestnol 136 (6) 144 (9) 137 (6) Cmpesterol 191 (10) 433 (22) 271 (20),f Sitosterol 165 (8) 205 (12) 204 (10) Sttistil nlysis: ANOVA followed y pirwise omprison y Student s t-test with Bonferroni orretion. Vlues re men (SEM). P ompred with olive oil. d P 0.05 ompred with olive oil nd rpeseed oil. e P 0.01 ompred with rpeseed oil. f P ompred with rpeseed oil. nd SO diets. In one report, high sturted ft intke (46 E%) (espeilly myristi nd plmiti id) ws ssoited with n inresed mss of lrge, holesterol-enrihed LDL prtiles (28). Although the differenes in the mount of myristi nd plmiti id in the OO diet (3 nd 19 mol%, respetively) ompred with the RO (4 nd 15 mol%, respetively) nd SO diets (4 nd 16 mol%, respetively) were smll it my hve ontriuted to the higher numer of the lrge nd medium-sized LDL sufrtions fter the OO diet oserved in this study. An lterntive explntion ould e different impt of the ftty id omponents on hepti lipse (HL) tivity. An inverse reltionship etween the onentrtion nd size of lrge, uoynt LDL prtiles nd HL tivity hs een reported (28, 50, 51). In hs een suggested tht n 3 ftty ids from fish oil ffet the omposition of lipoprotein sufrtions fvorly y lowering the TAG ontent (29), ut the interprettion of the omposition dt is not ler. A redued TAG ontent might e fvorle euse TAG-poor LDL prtiles re tolized fster thn TAG-rih prtiles (29). However, redution of the TAG ontent my lso result in redution of the size of the prtile, whih is not onsidered fvorle. Our dt showed tht RO onsumption resulted in higher numer of TAG prtiles in most LDL sufrtions ompred with OO nd SO without ffeting the size of the lipoproteins. In this study, HDL holesterol onentrtions tended to e higher fter OO nd RO ompred with SO (P 0.06). In ordne, Mt et l. (43) found lower HDL holesterol nd poa-i levels fter PUFA-rih diet ompred with MUFA-rih diet, while Crmen et l. (52) found lower HDL holesterol fter SO ompred with OO. Informtion out the effet of diet on HDL sulsses is limited. We oserved lower onentrtion of holesterol, TAG, nd PL in HDL 2 fter SO ompred with OO nd RO. The reltively high intke of n 6 PUFA during the SO diet (15 E%) my e the reson for the lower poa-i onentrtion in plsm nd HDL 2 oserved fter SO (53). In ontrst, Vlst et l. (54) oserved no differenes in HDL 2 or HDL 3 in helthy women nd men etween RO nd SO diets, while Dreon et l. (55) reported higher HDL 2 holesterol fter PUFA ompred with MUFA. An inrement in HDL 3 fter fish oil supplements hs een reported in hypertriglyeridemi ptients (56), while in type II hyperlipoproteinemi, n 3 ftty ids signifintly inresed HDL 2 without ltertions in HDL 3 (29). In this study the MUFA-rih diets ontining OO nd RO hd more fvorle effets on HDL sulsses thn the PUFArih diet ontining SO. The onventionl risk mrkers of IHD, plsm totl holesterol nd LDL holesterol, were signifintly higher fter 3 weeks of onsuming diet rih in OO ompred with RO nd SO. The oserved differene etween OO nd RO/SO is not surprising, euse similr results were otined in other humn experiments ompring OO nd RO (57) nd OO nd SO (52, 58). In ddition, it hs een shown tht n OO diet resulted in similr plsm holesterol onentrtions s plmolein diet rih in SFA (59, 60), while RO resulted in lower plsm holesterol thn plmolein (61). However, other omprisons of OO with RO or SO resulted in virtully similr lood lipid nd LDL holesterol onentrtions (62, 63). In ordne with our results, similr lood lipid onentrtions fter RO- nd SO-ontining diets hve een oserved in numer of other studies (53, 64 66). In this study the plsm holesterol differene etween OO nd RO ould e explined only prtly y differenes in ftty id omposition. Aording to lgorithms of Hegsted et l. (4) nd Keys, Anderson, nd Grnde (1) this study should result in differene in plsm totl holesterol of mm etween OO nd RO. The oserved differene ws lrger thn predited, nmely 0.5 mm, while the plsm holesterol differene etween OO nd SO ws virtully identil to the predited differene. OO is rih in squlene (3,651 mg/kg oil) ompred with RO (10 mg/kg oil) nd SO (134 mg/kg oil) (Tle 3). The higher intke of squlene during the OO diet resulted in signifintly higher plsm squlene:holesterol rtios (Tle 9). In ddition, the plsm desmosterol: holesterol rtio ws lso higher fter the OO diet ompred with RO nd SO diets, inditing tht higher level of holesterol synthesis took ple during the OO diet. This is likely to hve ontriuted to the higher holesterol onentrtion fter OO ompred with RO nd SO. In greement, Miettinen nd Vnhnen (67) found tht squlene hd holesterol-rising properties when 1 g of squlene ws dded to RO. Plnt sterols re sored to some extent (mpesterol, 10 14%; sitosterol, 1 5%) (68) nd the lower ontents of mpesterol nd sitosterol in OO ompred with RO nd SO (Tle 3) were refleted diretly in the plsm rtios of these sterols (Tle 9). Plnt sterols interfere with intestinl holesterol sorption (69) nd hve the ility to lower totl nd LDL holesterol (70). The plsm levels of mpesterol nd sitosterol in helthy sujets hve een found to e inversely relted to the overll holesterol synthesis (71). Beuse plsm rtios of mpesterol nd sitosterol to holesterol were lower fter the OO diet 1908 Journl of Lipid Reserh Volume 41, 2000

9 ompred with the RO nd SO diets, this my offer n dditionl explntion for the higher plsm nd LDL holesterol onentrtions oserved fter onsumption of the OO diet. The higher plsm TAG onentrtion fter OO thn fter RO nd SO oserved in this study hs een reported in some studies (43, 64), while numer of studies hve not found OO to e hypertriglyeridemi ompred with other oils (55, 63, 72). A met-nlysis designed to ompre effets of MUFA nd PUFA diets on lood lipids nd lipoproteins reported onsistently lower TAG onentrtions fter high PUFA diets (73). Thus, the lower ontent of PUFA in OO, ompred with RO nd SO, my ontriute to the higher TAG onentrtions fter OO. In ddition, if squlene enhnes the hepti holesterol ontent euse of n inresed holesterol synthesis, then the LDL reeptor tivity would e downregulted ffeting the removl of oth LDL, VLDL nd IDL prtiles. This might explin the oserved inrese in VLDL TAGs (nd totl TAGs) oserved in this study nd y others fter onsumption of OO/squlene (67). As hypothesized, this study resulted in higher LDL: HDL nd totl:hdl holesterol rtios fter the OO nd SO diets ompred with the RO diet. This is in ordne with Vlst et l. (53) who found more fvorle HDL 2 :LDL rtio fter RO ompred with SO, nd with Crmen et l. (52) who reported no differene in totl: HDL holesterol rtio etween OO nd SO. In ontrst, other studies hve reported no differenes in the LDL: HDL holesterol rtio etween RO nd SO (65, 74) nd onfliting results regrding the totl:hdl holesterol rtio when ompring OO nd SO (43, 58). The totl:hdl holesterol rtio hs een reported to e etter preditor of IHD thn other onventionl risk mrkers (totl holesterol, LDL holesterol or TAG) in numer of studies (75). Thus, in this study the RO diet resulted in the most fvorle lipid rtios ompred with oth OO nd SO. In ddition, ll other mesured onventionl risk mrkers of IHD (HDL holesterol, poa, LDL holesterol, pob) nd the LDL nd HDL sufrtions were more fvorly ffeted y RO nd SO ompred with OO. Thus, tken together, the RO diet seemed to hve more fvorle effets with regrd to lood lipid nd lipoprotein sufrtions ompred with SO nd RO. However, results from nlyses of ogultion ftors suggest tht OO hs n ntithromoti effet ompred with SO (nd RO) tht might ountert its less fvorle plsm lipid effets (31, 76). In onlusion, the results from this study suggest tht RO nd SO rih diets ompred with OO hve more fvorle effets on plsm lipid nd lipoprotein onentrtions in young helthy sujets. The differenes my in prt e ttriuted to differenes in the mount of nonftty id omponents of the oils. In ddition, when vegetle oils ounted for sustntil prt of the totl ft intke, the oil qulity ws shown to ffet the LDL nd HDL sulss profiles differently. Whether these differenes my e due to differenes in ftty id omposition is still to e resolved. The uthors would like to thnk Len T. Andersen, Kitt S. Hoffmn, Kir H. B. Lrsen, Klr Jørgensen, Kirsten B. Rsmussen, Siene Jotternd, Dgmr Reduth, nd Leen Kipiinen for exellent tehnil ssistne with this study. This study ws prt of the reserh progrm Rpeseed Oil in Humn Nutrition finned y the Dnish Food Tehnology Reserh Progrm (FØTEK-2/93s-2469-å ). Mnusript reeived 14 Mrh 2000 nd in revised form 10 July REFERENCES 1. Keys, A., J. T. Anderson, nd F. Grnde Serum holesterol response to hnges in the diet. IV. Prtiulr sturted ftty ids in the diet. Metolism. 14: Hegsted, D. M., R. B. MGndy, M. L. Myers, nd F. J. Stre Quntittive effets of dietry ft on serum holesterol in mn. Am. J. Clin. Nutr. 17: Mensink, R. P., nd M. B. Ktn Effet of dietry ftty ids on serum lipids nd lipoproteins. A met-nlysis of 27 trils. Arteriosler. Throm. 12: Hegsted, D. M., L. M. Ausmn, J. A. Johnson, nd G. E. Dlll Dietry ft nd serum lipids: n evlution of the experimentl dt. Am. J. Clin. Nutr. 57: Yu, S., J. Derr, T. D. Etherton, nd P. M. Kris Etherton Plsm holesterol-preditive equtions demonstrte tht steri id is neutrl nd monounsturted ftty ids re hypoholesterolemi. Am. J. Clin. Nutr. 61: Howell, W. H., D. J. MNmr, M. A. Tos, B. T. Smith, nd J. A. Gines Plsm lipid nd lipoprotein responses to dietry ft nd holesterol: met-nlysis. Am. J. Clin. Nutr. 65: Genest, J., Jr., J. R. MNmr, J. M. Ordovs, J. L. Jenner, S. R. Silermn, K. M. Anderson, P. W. Wilson, D. N. Slem, nd E. J. Shefer Lipoprotein holesterol, polipoprotein A-I nd B nd lipoprotein () normlities in men with premture oronry rtery disese. J. Am. Coll. Crdiol. 19: Despres, J. P., B. Lmrhe, P. Muriege, B. Cntin, G. R. Dgenis, S. Moorjni, nd P. J. Lupien Hyperinsulinemi s n independent risk ftor for ishemi hert disese. N. Engl. J. Med. 334: Lmrhe, B., A. Thernof, P. Muriege, B. Cntin, G. R. Dgenis, P. J. Lupien, nd J. P. Despres Fsting insulin nd polipoprotein B levels nd low-density lipoprotein prtile size s risk ftors for ishemi hert disese. J. Am. Med. Asso. 279: Hoknson, J. E., nd M. A. Austin Plsm triglyeride level is risk ftor for rdiovsulr disese independent of highdensity lipoprotein holesterol level: met-nlysis of popultionsed prospetive studies. J. Crdiovs. Risk. 3: Fryn, K. N., C. M. Willims, nd P. Arner Are inresed plsm non-esterified ftty id onentrtions risk mrker for oronry hert disese nd other hroni diseses? Clin. Si. 90: Kruss, R. M., F. T. Lindgren, P. T. Willims, S. F. Kelsey, J. Brensike, K. Vrnizn, K. M. Detre, nd R. I. Levy Intermeditedensity lipoproteins nd progression of oronry rtery disese in hyperholesterolemi men. Lnet. 2: Steiner, G., L. Shwrtz, S. Shumk, nd M. Popst The ssoition of inresed levels of intermedite-density lipoproteins with smoking nd with oronry rtery disese. Cirultion. 75: Mk, W. J., R. M. Kruss, nd H. N. Hodis Lipoprotein sulsses in the Monitored Atheroslerosis Regression Study (MARS). Tretment effets nd reltion to oronry ngiogrphi progression. Arteriosler. Throm. Vs. Biol. 16: Hodis, H. N., W. J. Mk, M. Dunn, C. Liu, R. H. Selzer, nd R. M. Kruss Intermedite-density lipoproteins nd progression of rotid rteril wll intim-medi thikness. Cirultion. 95: Bumstrk, M. W., A. Berg, M. Hlle, U. F. E. Rensing, H. Roskmm, nd J. Keul Smll, dense LDL re mjor determinnt for the serverity of ngiogrphilly ssessed CAD, even in groups with similr triglyeride levels. (strt). Atheroslerosis. 109: Austin, M. A., J. L. Breslow, C. H. Hennekens, J. E. Buring, W. C. Pedersen et l. Dietry oils nd lipoprotein sufrtions 1909

10 Willett, nd R. M. Kruss Low-density lipoprotein sulss ptterns nd risk of myordil infrtion. J. Am. Med. Asso. 260: Grdner, C. D., nd H. C. Kremer Monounsturted versus polyunsturted dietry ft nd serum lipids. A met-nlysis. Arteriosler. Throm. Vs. Biol. 15: Rjmn, I., M. J. Kendll, R. Crm, R. L. Holder, M. Slih, nd M. D. Gmmge Investigtion of low density lipoprotein sufrtions s oronry risk ftor in normotriglyeridemi men. Atheroslerosis. 125: Stmpfer, M. J., R. M. Kruss, J. M, P. J. Blnhe, L. G. Holl, F. M. Sks, nd C. H. Hennekens A prospetive study of triglyeride level, low-density lipoprotein prtile dimeter, nd risk of myordil infrtion. J. Am. Med. Asso. 276: Lmrhe, B., A. Thernof, S. Moorjni, B. Cntin, G. R. Dgenis, P. J. Lupien, nd J. P. Despres Smll, dense low-density lipoprotein prtiles s preditor of the risk of ishemi hert disese in men. Prospetive results from the Quee Crdiovsulr Study. Cirultion. 95: Nigon, F., P. Lesnik, M. Rouis, nd M. J. Chpmn Disrete suspeies of humn low density lipoproteins re heterogeneous in their intertion with the ellulr LDL reeptor. J. Lipid Res. 32: de Grf, J., H. L. M. Hk-Lemmers, M. P. Hetors, P. N. Demker, J. C. Hendriks, nd A. F. Stlenhoef Enhned suseptiility to in vitro oxidtion of the dense low density lipoprotein sufrtion in helthy sujets. Arteriosler. Throm. 11: Aner, V., J. S. Millr, M. MConnell, J. Shepherd, nd C. J. Pkrd Intertion of very-low-density, intermedite-density, nd low-density lipoproteins with humn rteril wll proteoglyns. Arteriosler. Throm. Vs. Biol. 17: Austin, M. A., M. C. King, K. M. Vrnizn, nd R. M. Kruss Atherogeni lipoprotein phenotype. A proposed geneti mrker for oronry hert disese risk. Cirultion. 82: Griffin, B. A Low-density lipoprotein sulsses: mehnisms of formtion nd modultion. Pro. Nutr. So. 56: Kruss, R. M., nd D. M. Dreon Low-density-lipoprotein sulsses nd response to low-ft diet in helthy men. Am. J. Clin. Nutr. 62: 478S 487S. 28. Dreon, D. M., H. A. Fernstrom, H. Cmpos, P. Blnhe, P. T. Willims, nd R. M. Kruss Chnge in dietry sturted ft intke is orrelted with hnge in mss of lrge low-density-lipoprotein prtiles in men. Am. J. Clin. Nutr. 67: Bumstrk, M. W., I. Frey, A. Berg, nd J. Keul Influene of n-3 ftty ids from fish oils on onentrtion of high- nd lowdensity lipoprotein sufrtions nd their lipid nd polipoprotein omposition. Clin. Biohem. 25: Tinker, L. F., E. J. Prks, S. R. Behr, B. O. Shneemn, nd P. A. Dvis (n-3) ftty id supplementtion in modertely hypertriglyeridemi dults hnges postprndil lipid nd polipoprotein B responses to stndrdized test mel. J. Nutr. 129: Lrsen, L. F., J. Jespersen, nd P. Mrkmnn Are olive oil diets ntithromoti? Diets enrihed with olive, rpeseed, or sunflower oil ffet postprndil ftor VII differently. Am. J. Clin. Nutr. 70: Kirsten, W. J., nd G. U. Hesselius Rpid, utomti, high pity Dums determintion of nitrogen. Mirohem. J. 28: Folh, J., M. Lees, nd G. H. S. Stnley A simple method for the isoltion nd purifition of totl lipides from niml tissues. J. Biol. Chem. 226: Asp, N. G., C. G. Johnsson, H. Hllmer, nd M. Siljeström Rpid enzymti ssy of insolule nd solule dietry fier. J. Agri. Food Chem. 31: Morrison, W. R., nd L. M. Smith Preprtion of ftty id methyl esters nd dimethyletls from lipids with oron fluoridemethnol. J. Lipid Res. 5: Miettinen, T. A Cholesterol metolism during ketoonzole tretment in mn. J. Lipid Res. 29: Lindgren, F. T., L. L. Jensen, nd F. T. Hth The isoltion nd quntittive nlysis of serum lipoproteins. In Blood Lipids nd Lipoproteins: Quntittion, Composition nd Metolism. G. J. Nelson, editor. New York: Wiley-Intersiene Lindgren, F. T Preprtive ultrentrifugl lortory proedures nd suggestions for lipoprotein nlysis. In Anlysis of Lipids nd Lipoproteins. E. G. Perkins, editor. Chmpign, IL: Amerin Oil Chemists Soiety Anderson, D. W., A. V. Nihols, T. M. Forte, nd F. T. Lindgren Prtile distriution of humn serum high density lipoproteins. Biohim. Biophys. At. 493: Anderson, D. W., A. V. Nihols, S. S. Pn, nd F. T. Lindgren High density lipoprotein distriution. Resolution nd determintion of three mjor omponents in norml popultion smple. Atheroslerosis. 29: Bumstrk, M. W., W. Kreutz, A. Berg, I. Frey, nd J. Keul Struture of humn low-density lipoprotein sufrtions, determined y X-ry smll-ngle sttering. Biohim. Biophys. At. 1037: Altmn, D. G Clinil trils. In Prtil Sttistis for Medil Reserh. London: Chpmn & Hll Mt, P., L. A. Alvrez Sl, M. J. Ruo, J. Nuno, nd M. De Oy Effets of long-term monounsturted- vs polyunsturtedenrihed diets on lipoproteins in helthy men nd women. Am. J. Clin. Nutr. 55: Appleyrd, M The Copenhgen City Hert Study Østerroundersøgelsen. Snd. J. So. Med. Suppl. 41: Sndström, B., P. Mrkmnn, nd N. Bindslev An eightmonth ontrolled study of low-ft high-fire diet: effets on lood lipids nd lood pressure in helthy young sujets. Eur. J. Clin. Nutr. 46: Tholstrup, T., P. Mrkmnn, J. Jespersen, nd B. Sndström Ft high in steri id fvorly ffets lood lipids nd ftor VII ogulnt tivity in omprison with fts high in plmiti id or high in myristi nd luri id. Am. J. Clin. Nutr. 59: Cmpos, H., K. S. Arnold, M. E. Blestr, T. L. Innerrity, nd R. M. Kruss Differenes in reeptor inding of LDL sufrtions. Arteriosler. Throm. Vs. Biol. 16: Ptsh, W., R. Ostlund, I. Kuisk, R. Levy, nd G. Shonfeld Chrteriztion of lipoprotein in kindred with fmilil hyperholesterolemi. J. Lipid Res. 23: Cmpos, H., G. O. Roederer, S. Lussier Cn, J. Dvignon, nd R. M. Kruss Predominne of lrge LDL nd redued HDL 2 holesterol in normolipidemi men with oronry rtery disese. Arteriosler. Throm. Vs. Biol. 15: Zmon, A., M. A. Austin, B. G. Brown, J. E. Hoknson, nd J. D. Brunzell Effet of hepti lipse on LDL in norml men nd those with oronry rtery disese. Arteriosler. Throm. 13: Wtson, T. D., M. J. Cslke, D. J. Freemn, B. A. Griffin, J. Hinnie, C. J. Pkrd, nd J. Shepherd Determinnts of LDL sufrtion distriution nd onentrtions in young normolipidemi sujets. Arteriosler. Throm. 14: Crmen, R., J. F. Asso, G. Cmejo, G. Vrel, E. Hurt Cmejo, J. M. Ordovs, J. Mrtinez Vlls, M. Bergstom, nd B. Wllin Effet of olive nd sunflower oils on low density lipoprotein level, omposition, size, oxidtion nd intertion with rteril proteoglyns. Atheroslerosis. 125: Vlst, L. M., M. Juhiinen, A. Aro, M. B. Ktn, nd M. Mutnen Effets of monounsturted rpeseed oil nd polyunsturted sunflower oil diet on lipoprotein levels in humns. Arteriosler. Throm. 12: Shepherd, J., C. J. Pkrd, J. R. Ptsh, A. M. Gotto, Jr., nd O. D. Tunton Effets of dietry polyunsturted nd sturted ft on the properties of high density lipoproteins nd the metolism of polipoprotein A-1. J. Clin. Invest. 61: Dreon, D. M., K. M. Vrnizn, R. M. Kruss, M. A. Austin, nd P. D. Wood The effets of polyunsturted ft vs monounsturted ft on plsm lipoproteins. J. Am. Med. Asso. 263: Dek, C., nd K. Rdk Effets of modest doses of omeg-3 ftty ids on lipids nd lipoproteins in hypertriglyeridemi sujets. A rndomized ontrolled tril. Arh. Intern. Med. 149: Lihtenstein, A. H., L. M. Ausmn, W. Crrso, J. L. Jenner, L. J. Gultieri, B. R. Goldin, J. M. Ordovs, nd E. J. Shefer Effets of nol, orn nd olive oils on fsting nd postprndil plsm lipoproteins in humns s prt of Ntionl Cholesterol Edution Progrm Step 2 Diet. Arteriosler. Throm. 13: Jimenez, P. F., A. Espino, F. Lopez Segur, J. Blno, V. Ruiz Gutierrez, J. L. Prd, J. Lopez Mirnd, J. Jimenez Pereperez, nd J. M. Ordovs Lipoprotein onentrtions in normolipidemi mles 1910 Journl of Lipid Reserh Volume 41, 2000