Factors influencing Lp[a]- particle size as determined by gradient gel electrophoresis

Transcription

1 Fctors influencing Lp[]- prticle size s determined by grdient gel electrophoresis Dvid O'Nel,*>t Grhme Grieve,**t Derek Re,t George Drgicevic,' nd Jmes D. Best19'9t Deprtment of Medicine,* The University of Melbourne, nd Deprtment of Clinicl Biochemistry: St. Vincent's Hospitl, Fitzroy, Austrli 3065 Abstrct This study exmined fctors influencing the prticle dimeter of Lp[]-, the low density lipoprotein (LDL)-like moiety of Lp[], in 26 subjects chosen to provide rnge of Lp[] nd triglyceride levels. Lp[] nd LDL frctions were isolted by verticl density ultrcentrifugtion. Lp[] ws further purified using IysineSephrose ffinity column nd Lp[]- obtined by incubting Lp[] with dithiothreitol. Lp[], LDL, nd Lp[]- frctions were run on 3-13% grdient gels to determine prticle dimeter. Lp[] size correlted positively with LDL size (r = 0.62; P < 0.001), but the ssocition between Lp[]- size nd LDL size ws stronger (r = 0.82; P < ). Log triglyceride level correlted inversely with Lp[]- size (r = -0.72; P < ) nd LDL size (r = -0.69; P < ). HDL cholesterol level correlted positively with Lp[]- size (r = 0.67; P < ) nd LDL size (r = 0.64; P < 0,0005). M The strong correltion between LDL size nd Lp[]- size my be due to extrcellulr utiliztion of circulting LDL in the production of Lp[] or my reflect the sme metbolic processes influencing both these prticles once Lp[] hs been formed.-@nel, D., C. Grieve, D. Re, G. Drgicevic, nd J. D. Best. Fctors influencing Lp[]- prticle size s determined by grdient gel e1ectrophoresis.j. Lipid Res : Supplementry key words lipoprotein[] lipoprotein[]- low density lipoproteins First described by Berg in 1963 (l), lipoprotein[] or Lp[] consists of glycoprotein po[] linked to polipoprotein B-100 (pob-100) on n LDL-like prticle Lp[]- (2). Both po[] nd pob-100 moieties re synthesized primrily within heptocytes (3, 4) though it remins uncertin s to where they re ssembled to form Lp[]. Lp[] hs been reported to be risk fctor for ischemic hert disese (5-), cerebrovsculr disese (9, lo), nd peripherl vsculr disese (11). Bsed on the number of repet sequences of cysteine-rich, internlly looped structures known s kringles, po[] cn be seprted into different isoforms (12). Concentrtion of Lp[] hs been shown to vry inversely with po[] isoform size (13-16). Consequently, epidemiologicl studies of the contribution of Lp[J to crdiovsculr risk hve gener- lly focused their ttention upon the po[] moiety, with Lp[]- being reltively ignored (5-1 1). Our im ws to determine fctors influencing the prticle dimeter of Lp[] nd, in prticulr, of Lp[]-, the LDLlike moiety of Lp[] tht is depleted of po[]. The reltionship of the size of Lp[]- to LDL prticle size ws of mjor interest. The structurl similrity between Lp[]- nd LDL rises the possibility tht there my be inter-individul vrition in the prticle size of Lp[]-, heterogeneity tht hs been extensively documented for LDL prticles (1 7-19). We lso wnted to investigte the reltive effects of metbolic influences on ech of these lipoprotein clsses. Previous studies hve shown tht there is strong inverse correltion between the dimeter of the LDL prticle nd the degree of hypertriglyceridemi within n individul (19-2 1). Size heterogeneity of Lp[]- prticles is of potentil clinicl relevnce s Austin et l. (21,22) hve shown tht LDL prticle size, determined by nondenturing grdient gel electrophoresis, is relted to therogenic risk. Smller, more dense prticles re thought to confer n incresed risk. By convention, Phenotype A refers to those individuls with n LDL prticle size distribution contining predominntly lrger prticles with pek size nm, while Phenotype B refers to those individuls with n LDL prticle size distribution contining predominntly smller prticles ((25.5 nm). Recent electron cryomicroscopy studies hve indicted tht Lp[] prticles re roughly sphericl in shpe (23), which would mke nondenturing grdient gel electrophoresis suitble method by which to determine their size. If Lp[]- prticle size displyed n inter-individul vrition simi- Abbrevitions: LDL, low density lipoprotein; Lp[], lipoprotein[]; Lp[]-, LDL-like moiety of Lp[]; po, polipoprotein; CV, coefficient of vrition; HDL, high density lipoprotein; TBS, Tris-buffered sline; PBS, phosphte-buffered sline; DlT. dithiothreitol. 'To whom correspondence should be ddressed. Journl of Lipid Reserch Volume 37,

2 lr to tht described for LDL, then smll dense Lp[]- could hve prticulr importnce in the genesis of therosclerosis. MATERIALS AND METHODS I1 dibetes mellitus. All subjects were cliniclly stble without ny intercurrent illness. None of the subjects ws receiving &blockers, lipid modifying mediction, estrogen, or corticosteroid therpy. Informed consent ws obtined nd the study ws pproved by the St. Vincent's Hospitl Humn Reserch Ethics Committee. Subjects Twenty-six subjects (17 mle/ 9 femle, ged 57 k 3 yers) were selected from the stff nd outptient popultion of St. Vincent's Hospitl. As there is structurl nd biochemicl similrity between Lp[]- nd LDL, nd in the light of the reported reltionship between LDL size nd triglyceride level, the subjects were chosen to disply the effect of brod rnge of triglyceride levels on Lp[] nd Lpfl- size (Tble 1). A wide rnge of po[] levels ws lso desirble in order to demonstrte the effect of po[] on Lpf] prticle size. Sixteen subjects were norml volunteers nd 11 subjects hd Type Lipoprotein nlysis Ten ml of blood ws collected in n EDTA-contining tube from ll subjects, fter 12-h overnight fst. Plsm ws seprted by centrifugtion t 1300 g for 15 min t 4 C. Totl plsm cholesterol (cholesterol oxidse, Trce Scientific, Melbourne, Austrli) nd totl plsm triglyceride (glycerol-3-phosphte oxidse, Trce Scientific, Melbourne, Austrli) determintions were performed s prt of routine utomted lbortory enzymtic ssy (24) on n Olympus AU5031. The interssy coefficient of vrition (CV) for cholesterol ws 1.6% nd 0.9% t 3.2 mmol/l nd 7.8 mmol/l, respectively. TABLE 1. Apo[] level, Lp[] size, clculted contribution of po[] to Lp[] size, po[] phenotype, nd triglyceride levels of individul subiects who re identified in order of incresine Dorl level Subject Apo[] Level Lp[] Size Lp[] - (Lp[]-l Size Apo[] Phenotype Triglyceride nm nm mmol/l >s4/>s >s4/>s s >s4/>s >s4/s >s4/s >s4/s Sl/B Sl/S >s4/s >s4/s ,s S4/F Sl/S3 1.o Sl/B s Sl/S s s s s s s Sl/S Sl/S Sl/S Journl of Lipid Reserch Volume 37, 1996

3 For triglycerides, the CV ws 1.9% t 1.0 mmol/l nd 1.8% t 2.2 mmol/l. I-IDL cholesterol ws ssyed fter polyethylene glycol 6000 precipittion of non-hdl lipoproteins (25). CV ws 5.5% t 2.03 mmol/l nd 4% t 0.97 mmol/l. LDL cholesterol ws clculted using the eqution of Friedewld, Levy, nd Fredrickson (26) when the totl triglyceride level ws 54.5 mmol/l. For higher levels, VLDL ws isolted by ultrcentrifugtion nd cholesterol ws mesured seprtely. In these cses LDL cholesterol ws clculted by subtrction of HDL nd VLDL cholesterol from totl cholesterol levels. Assy of po[] levels ws performed by IRMA (27) using commercil kit (Phrmci, Uppsl, Sweden). Interssy CV ws 14% t 112 U/1 nd 9% t 380 U/1. Identifiction of Lp[] bnds Vlidtion studies were performed using plsm from four of the subjects. Preprtion of Lp[] ws bsed on n dpttion of the method of Chung et l. (28). After 12-h fst, 10 ml of whole blood ws collected from subjects in n EDTAcontining tube nd ws centrifuged t 1500 g for min to seprte the plsm. The density of 4 ml of the plsm ws djusted to 1.21 g/ml by the ddition of 1.31 g of KBr. This plsm ws then pipetted into n ultrcentrifuge tube nd overlyered with g/ml solution. The tubes were seled nd plced in Beckmn VTi65.1 rotor nd spun for 90 min t rpm t 7 C with ccelertion nd decelertion prmeters set t 6. After ultrcentrifugtion, LDL formed visible bnd in the middle of the centrifuge tube with HDL remining t the bottom of the tube. The tubes were then frctionted using frctiontor nd frction collector (ISCO Lincoln, NB). The ultrcentrifuge tubes were punctured t their bse without disturbing the LDL lyer nd dense immiscible liquid (Mxidens, Nycomed, Oslo, Norwy) ws pumped by syringe pump (Rzel, Stnford, CT) into the bse of the tube displcing its contents upwrds. The frction collector ws progrmmed to collect 13 drops per frction (400 pl). Apo[] nd cholesterol levels were ssyed on ll frctions to identify the density of Lp[] prticles in the ultrcentrifuge tube reltive to LDL nd HDL prticles. In ddition, ll frctions were subjected to non-denturing grdient gel electrophoresis. The method used ws modified form of tht described by Kruss nd Burke (18). Approximtely 80 p1 ws dded to 20 p1 of trcking dye nd 60 pl of Tris-borte buffer (ph 8.4) nd run t 180 volts for 12 h on 2-9% non-denturing polycrylmide grdient gel. All frctions were run in duplicte on identicl gels, with one stined for protein nd the other immunoblotted with ntibody to po[]. This step ws done to identify the Lp[] bnds nd their reltive positions on the gels. The gels stined for protein were first fnced with 10% 5-sulfoslicylic cid solution for 1 h nd then stined with Coomssie blue by immersion in stin for 3-4 h. Next, the gels were destined in 5% cetic cid solution for h. Western blotting (29) ws performed on the corresponding gels by the trnsfer of proteins to nitrocellulose membrne (0.45 nm pore size) using Bio-Rd semidry electroblotter for 50 min t 15 volts. The membrne ws immersed in 5% skimmed milk powder solution for 1 h t room temperture to block unbound sites. The membrne ws then incubted overnight in 5% skimmed milk solution in TBS (Tris-buffered sline: 20 mm Tris, 50 mm NCI, ph 7.5) contining 1:500 dilution of mouse monoclonl ntibody ginst po[]. The next morning, fter extensive wshes with TBS nd Tween TBS (0.05% Tween 20 detergent in TBS), the membrne ws incubted for t lest 2 h with the secondry ntibody. After further wshes with Tween TBS nd TBS, the membrne ws plced into color development solution contining the substrte 5-bromo4chloro-3-indoyl-phosphte nd nitroblue tetrzolium until the bnds developed. Sizing of Lp[], Lp[]-, nd LDL Lp[] ws initilly isolted s described bove using single verticl spin density grdient ultrcentrifugtion with spirtion of the Lp[] frction. We hd previously estblished tht the Lp[] pek ws situted between LDL nd HDL (see below), usully not visible to the nked eye. In some subjects where the po[] level ws greter thn U/1, the Lp[] frction becme visible, first s fint bnd which then becme progressively stronger with incresing levels. Becuse of significnt contmintion of the spirted Lp[] with LDL, further purifiction step ws necessry nd ws chieved using lysineephrose ffhity column. The spirted smple ws injected onto Phrmci XK 500 column contining 15 g of lysine- Sephrose equilibrted in phosphte-buffered sline (PBS). Elution of ny contminting LDL nd slts ws chieved by wshing the column out with PBS. After the contminnts were removed, the Lp[] ws then eluted with 5.3 g/l of E-minocproic cid in PBS. The eluent ws monitored t both 234 nm, which detects slts, lipid nd protein nd gve greter sensitivity for detection of the Lp[] pek, nd 280 nm to detect predominntly protein (Fig. 1). The Lp[] pek ws collected nd then concentrted by dilysis ginst polyethylene glycol 8000 in 10,000 MW cut-off filter resulting in n p proximtely 20-fold concentrtion. In subjects with po[] levels greter thn 1000 U/ml, 4 ml of plsm yielded sufficient mount of Lp[] to permit sizing, but in those subjects with po[] levels less thn 100 U/ml, up to 44 ml of plsm ws required. O'Nel et l. Fctors influencing Lp[]-prticle size 1657

4 time (Kcs.) Fig. 1. Elution profile from the IysineSephrose ffinity column, demonstrting seprtion of LDL from Lp[] in subject 17, whose po[] level ws 1030 U/I (see Tble 1). Absorbnces were set t () 234 nm, which detects slts, lipid nd protein nd gve greter sensitivity for detection of the Lp[] pek, nd (b) 280 nm, which detects predominntly protein. The initil lrge pek indictes the elution of slts nd LDL. Subsequently Lp[] ws eluted with &-mine cproic cid. The rrow indictes the beginning of the Lp[] pek. Lp[]- ws generted by incubting the isolted Lp[] with 0.01 M dithiothreitol (DIT) t room temperture for 1 h (30), thus cleving the disulphide link between pob-100 nd po[]. LDL ws isolted by the sme ultrcentrifugtion technique (28) used for isoltion of Lp[]. After ultrcentrifugtion, the LDL ws visible s bnd in the middle of the ultrcentrifuge tube, which ws spirted by direct puncture. Lp[], Lp[]-, nd LDL prticle dimeters were determined s previously described (18, 31) using commer- cilly vilble 3-13% nondenturing ntive gels (Grdipore, Sydney, Austrli). All gels used in this study were from the sme production run. Mrkers used were 29 nm ltex beds (Duke, Plo Alto, CA) nd Phrmci high moleculr weight stndrds (Phrmci, Pisctwy, NJ). The gels were scnned by Trcktel Video Densitometer Scnner (Vision System Ltd., Adelide, Austrli) to provide quntittive mesurement of the size of the pek nd its distnce from the origin. Prticle dimeter ws obtined by plotting the log of the known dimeter of the stndrds (y xis) ginst their positions on the scnned gel (x xis). A sttisticl pckge ws used to plot regression nd derive formul llowing unknown smples to be sized. Coefficient of vrition on 26.1 nm qulity control smple run on every gel ws 0.8%. To estblish tht the 1ysineSephrose column did not lter Lp[] prticle size, we selected two individuls with Lp[] levels high enough for bnd to be visible fter ultrcentrifugtion. This bnd ws spirted nd n liquot ws removed before further purifiction by the column. Both the direct spirte nd the smple run through the column for ech subject were run in djcent lnes on 3-13% grdient gel nd the size ws clculted. It ws lso importnt to estblish tht Lp[]- size ws not significntly ltered by the incubtion with DIT. Becuse of the similrity of LDL nd Lp[]-, this question ws tested by running 21 smples of LDL with nd without incubtion with DTT in djcent lnes on sizing gels. Apo[] phenotyping Fifteen to thirty p1 of plsm in EDTA ws used to determine po[] phenotype by estblished methods (12, 15). Isoform stndrds used were designted B, S1, S3, S4, >S4 (Lp[] phenotyping kit, Immuno-Ag, Vienn, Austri). Both smples nd stndrds were mixed with reducing buffer, incubted t 100 C for 5 min, cooled, Fig. 2. Cholesterol nd po[] levels mesured on the frctions collected from subject 17 (totl cholesterol 5.9 mmol/l, totl triglycerides 1.1 mmol/l, HDL cholesterol 0.97 mmov1, po[] 1,030 U/l). The cholesterol pek between frctions 8 nd 16 represents LDL cholesterol nd between frctions 21 nd 27 represents HDL cholesterol. The po[] pek is seen between HDL nd LDL peks. ~.ri~y@/d) Journl of Lipid Reserch Volume 37, 1996

5 nd run t 20 ma for 6 h t 4 C on 1.5% grose-sds gel in Bio-Rd-Protein I1 tnk. Resultnt bnds were blotted by cpillry ction onto nitrocellulose membrne; nonspecific binding sites were blocked by incubting the membrne for 60 min in blocking solution. The membrne ws then incubted with mouse monoclonl nti-po[] primry ntibody (kindly provided by Sntic Mrcovin, Northwest Lipid Reserch Lbortory, University of Wshington, Settle, WA) t 1:500 dilution for 6 h. After wshing, the membrne ws incubted with the secondry ntibody, GAM-Alkline phosphtse conjugte (Bio-Rd immunoblot kit C/N ), for 2 h. After further wshes the membrne ws plced into color development solution until the bnds developed. The po[] bnds tht developed were ssessed for homogeneity or heterogeneity nd migrtion compred with the stndrds. Sttisticl nlysis Sttisticl nlysis ws performed with Sttview (Abcus Concepts, Berkeley, CA) sttisticl pckge on Mcintosh computer. An nlysis of vrince (ANOVA) ws performed compring the sex of the subjects with ll the lipid prmeters studied. A simple liner regression nlysis ws used to compre the dimeter of lipid prticles exmined with ech other nd with the lipid levels mesured in plsm. Log-trnsformed po[] nd triglyceride levels were used in the sttisticl nlysis to normlize the distribution of ech prmeter. The other prmeters were included without modifiction. A pired Student's t test ws used to compre LDL prticle dimeter with tht of Lp[]- nd LDL dimeter fter incubtion with DlT. Results re given s men k SEM. RESULTS Identifiction of Lp[] bnd In ll four subjects, pek po[] levels ssyed in the ultrcentrifuged frctions were between the LDL nd HDL cholesterol peks in the density rnge g/ml (Fig. 2). On the grdient gels stined with Coomssie blue, frctions in which the po[] levels peked produced dominnt well-defined bnd tht did not trvel s fr into the gel nd hence hd greter dimeter thn LDL. This bnd ws confirmed s Lp[] by immunoblot (Fig. 3). Subject 17, whose immunoblot is shown, ws heterozygote with the phenotype Sl/S3 (Tble 1). A dominnt bnd is shown, but in ddition more fint bnd ws noted on immunoblot (see frctions 16 nd 17 in Fig. 3). Although it hs been reported tht po[] cn lso be ssocited with triglyceride-rich prticles in nonfsting subjects with hypertriglyceridemi (32), our results re in keeping with previous findings tht most of this polipoprotein is ssocited with n LDL-like prticle in the fsting stte (2). Sizing of Lp[], Lp[]-, nd LDL There ws no significnt effect of DTT on LDL size with men prticle dimeter in the DlT-treted LDL of 25.8 (ko.2)nm nd for the ntive LDL of 25.8 (*0.2)nm (r = 0.99; P < ). In the two individuls where Lp[] spirted directly ws run beside Lp[] eluted from the lysine-sephrose column on grdient gels, no differences were seen fter protein stining nd sizing (results not shown). One dominnt Lp[] bnd nd, fter incubtion with DlT, one dominnt Lp[]- bnd (Fig. 4) were observed in ll but one of the subjects. In subject 25 (Tble 1) two co-dominnt bnds were seen (Fig. 5) nd the Lp[] prticle size used in this cse represented the men of the two peks. The prticle sizes of both Lp[] nd Lp[]- displyed considerble heterogeneity mong different individuls. There ws no ssocition of Lp[] or Lp[]- size with either the sex or the ge of the subjects. A significnt correltion between LDL prticle size nd Lp[] prticle size (Fig. 6) ws noted (r = 0.62; P < 0.001). With the removl of the po[] moiety nd correltion of LDL prticle size with Lp[]- prticle size, this reltionship ws strengthened considerbly (r = 0.82; P < ). There ws, however, smll but significnt difference in the men size of LDL nd Lp[]- with Lp[]- being lrger thn LDL (26.1 f 0.1 nm vs f 0.1 nm; P < 0.05). There ws no ssocition between log po[] level nd Lp[]- size (P = 0.7). Apo[] lso contributed to Lp[] size, s shown by the inverse reltionship (r = -0.44; P < 0.05) between Lp[] size nd log po[] level using simple liner regression. A further mesure of the contribution of po[] to Lp[] prticle size ws derived by subtrcting Lp[]- size from Lp[] size to give Lp[] - {Lp[]-) size. This nlysis reveled significnt inverse reltionship between the log of the po[] level nd Lp[] - {Lp[]-) size (r= -0.55; P < 0.005). Tble 1 shows po[] level, triglyceride level, Lp[] dimeter, Lp[] - {Lp[]-) dimeter, nd po[] phenotype in ech of the 26 subjects studied. A significnt negtive ssocition of LDL prticle size with log triglyceride level (r = -0.69; P < ) nd positive ssocition with HDL cholesterol level (r = 0.64; P < ) ws seen. Similr ssocitions were noted for Lp[] nd Lpll-. Figure 6 demonstrtes the ssocition of Lp[]- size with totl triglyceride nd HDL cholesterol levels. There ws significnt inverse ssocition (r = -0.52; P < 0.01) between Lp[] dimeter nd log triglyceride level which ws strengthened fter the clevge of the po[] moiety by DTT nd formtion of Lp[]- (r = -0.72; P < ). A positive correltion (r = 0.44; P < 0.05) ws lso noted between HDL cholesterol nd O'Nel et l. Fctors influencing Lp[]-prticle size 1659

6 ES EG A2 AI FRACTION C FRACTION E! E( A A b FRACllON d F R ACT ION Fig. 3. Polycrylmide grdient gels (2-9%) on which frctions collected from subject 17 (see Fig. 2) were run. () Immunoblot with po[] ntibody of gel on which frctions 1-15 were run. (b) Immunoblot with po[] ntibody of gel on which frctions were run. (FS) indictes the position of the bse of the well in the stcking gel. (EG) indictes the interfce between the stcking nd grdient gels. (AI) indictes the dominnt Lp[] bnd detected by the blot. In ddition, second, finter, bnd is seen representing lrger prticle (A2).(c) Protein stin of gel on which frctions 1-15 from the sme subject were run. (d) Protein stin of gel on which frctions from the sme subject were run. (A) indictes tht only single Lp[] bnd ws seen in the protein stined gels. The position of the Lp[] bnd on the protein stin ws identicl to tht of the dominnt Lp[] bnd seen in the immunoblot. Lp[] size. As with triglyceride level, this reltionship ws strengthened (r = 0.67; P < ) with removl of the po[] moiety to produce Lp[]-. DISCUSSION In group of subjects with wide rnge of po[] levels, we hve isolted nd sized Lp[], demonstrting lipoprotein prticle tht hs both greter density nd size thn LDL. The density rnge in which pek Lp[] levels were detected in our study is consistent with the g/ml reported by previous investigtors (33, 34). There ws considerble size heterogeneity of Lp[] s reported previously by McNmr et l. (35), who first mde use of nondenturing polycrylmide grdient gels to identify individuls with elevted Lp[] levels. In this study we hve extended the technique further by incorporting stndrds tht hve llowed us to ssign prticle dimeter to Lp[] nd we hve used this technique to determine the size of the LDL-like moiety of Lp[] known s Lp[]-. Our dt indicte tht Lp[]- is smller in size thn Lp[] nd tht there is very close correltion between LDL nd Lp[]- prticle dimeter, finding tht invites comprison of these two cholesterol- nd pob-100-rich prticles. It hs been known for some time tht elevted triglyceride levels nd decresed HDL cholesterol levels, or fctors tht determine these levels, correlte with smller LDL prticle size nd tht smller, more dense LDL prticles re ssocited with n incresed risk of therosclerosis (20, 2 1). Our findings confirm this reltionship between lipid prmeters nd LDL size. In ddition, it is now pprent tht triglyceride nd HDL cholesterol levels, or fctors tht determine these levels, my lso ply n importnt role in determining Lp[] 1660 Journl of Lipid Reserch Volume 37, 1996

7 ?--- - Thy Fer - "1 Fig. 4. () Photogrph of 3-13% grdient gel showing qulity control (QC). high moleculr weight stndrds (thyroglobulin nd ferritin), 29 nm beds, Lp[], Lp[] treted with D7T to produce Lp[]-, nd LDL bnds of subject 1. (b) Trcings tken from densitometric scns of Lp[], Lp[]-, nd LDL bnds. I nci () 534 (b) LPbI iz - n LPbI- - 1 Fig. 5. () Photogrph of 3-13% grdient gel showing Lp[], Lp[] treted with D'IT to produce Lp[]-, nd LDL bnds of subject 25. (b) Trcings tken from densitometric scns of Lp[], Lp[]-, nd LDL bnds. the contribution heterogeneity of po[] mkes to vrition in Lp[] size. The study of Rinwter et l. (36) in lrge number of ptients hs estblished the dominnt contribution of po[] phenotype to Lp[] density. Our dt indicte the contribution of po[] to Lp[] size by mesuring Lp[] nd Lp[]- prticle sizes nd relting the difference in size to po[] level nd phenotype. The results indicte tht both po[] nd Lp[]- moieties mke independent contributions to Lp[] dimeter. Thus it ppers tht the sme metbolic fctors tht determine LDL size lso influence Lp[]-. This influence my occur prior to the ssocition of po[] nd Lp[]-, with the ltter being drwn from the pool of circulting LDL. Support for this mechnism comes from results of studies with line of mice expressing the humn po[] gene, which suggest tht humn po[] my be secreted into the plsm free of lipoprotein nd subsequently ssocite with LDL in the circultion (38). In ddition, dt from White, Rinwter, nd Lnford (39) indicte tht bboon heptocytes secrete po[] in the free form nd tht ssocition between po[] nd pob-100 occurs fter secretion. However, it lso remins possible tht the Lp[]- moiety is not drwn from the circulting LDL pool but, once formed, circulting Lp[] is subject to modifiction by the sme metbolic influences tht determine LDL prticle size. A hypothesis tht rises from this study is tht the therogenic risk ssocited with Lp[] my not be determined by the po[] level lone but lso by the nture O'Nel et l. Fctors influencing Lp[]prticle size 1661 size through n influence on the size of the Lp[]moiety. Rinwter et l. (36), using density grdient ultrcentrifugtion, hve reported tht po[] isoform ccounted for pproximtely 80% of vrition in Lp[] density. Residul Lp[] density (presumbly relted to the Lp[]- moiety) correlted with LDL density, HDL size, nd triglyceride level. The uthors concluded tht smll, dense Lp[] prticles re found under conditions leding lso to smll, dense LDL prticles nd to smll, dense HDL prticles. Our findings of strong correltion between Lp[]- prticle size nd LDL prticle size, s well s with HDL cholesterol level nd the inverse of log triglycerides, re entirely consistent with this recently published study. In n erlier study, Fless, ZumMllen, nd Scnu (37) used nlyticl equilibrium ultrcentrifugtion to exmine the reltive properties of Lp[], Lp[]-, nd LDL prticles. They found tht the moleculr weight of Lp[]ws pproximtely 10% greter thn LDL in the two subjects studied. Using the moleculr weight nd density of LDL nd Lp[]- of the two subjects from tht study nd mking the ssumption tht these prticles re sphericl, the clculted difference in dimeter is of the order of 2% in both subjects. We lso found tht the men dimeter of Lp[]- in our subjects ws greter thn the dimeter of LDL, with mximum difference of 2.3%nd men difference of 0.8%. Although the focus of this investigtion ws directed t the Lp[]- portion of Lp[], we hve lso ddressed

8 () 27*5] 27 r-0.62; P<O.OOl GJ Wl dzdm) Lptrl- " , * rize(nm) g -1.2: 1' r-o.61;p< /,+* -- ~W Fig. 6. () Plot of LDL prticle size ginst Lp[] prticle size. (b) Plot of LDL prticle size ginst Lp[]- prticle size. (c) Plot of log triglyceride level ginst Lp[]- prticle size. (d) Plot of HDL cholesterol ginst Lp[]- prticle size. of the Lp[]- moiety. We suggest tht in hypertriglyceridemic individuls, smll dense Lp[] prticles my increse crdiovsculr risk in mnner nlogous to tht described for smll dense LDL prticles (22). Those t prticulr risk would include ptients with Type I1 dibetes (40) nd ptients with renl filure treted by peritonel dilysis (31, 41). Thus individuls my be t greter risk of vsculr disese if their po[] glycoprotein is ssocited with smll dense Lp[]- prticles compred with other individuls whose po[] is ssocited with lrger, less dense Lp[]- prticles. With reference to this lst point, it is of interest tht severl previous studies hve filed to show n ssocition between po[] levels nd therosclerotic vsculr disese in dibetic ptients (42,43) where smll dense LDL nd presumbly smll dense Lp[] prticles predominte.l Dvid O'Nel ws the recipient of Merck, Shrp nd Dohme Fellowship in Lipid Reserch. The biochemistry lbortory stff t St. Vincent's Hospitl performed the lipid ssys. We thnk Alici Jenkins, Ken Sikris, nd Nichols Blsz for their vluble dvice. Mnuscript received 8 November 1995 nd in revised form 1 Mrch REFERENCES 1. Berg, K A new serum type system in mn-the Lp system. Act Pthol. Micmbiol. Scnd. 59: Utermnn, G The mysteries of lipoprotein[]. Science. 246: Krempler, F., G. M. Kostner, K. Bolzno, nd F. Sndhofer Turnover of lipoprotein [] in mn.j. CZin. Invest. 65: Hvel, R. J., nd J. P. Kne. Structure nd metbolism of lipoproteins In The Metbolic Bsis of Inherited Disese. 6th ed. C. R. Scriver, A. L. Beudet, W. S. Sly, nd D. Vlle, editors. McGrw-Hill, New York Renninger, W., G. G. Wendtt, P. Nwrocki, nd H. Weignd. 1965; Beitrg zur problemtik des Lp-systems. Humngenetik. 1: Berg, K., G. M. Dhlen, nd M. H. Frick Lipoprotein [] nd pre-1-lipoprotein in ptients with coronry hert disese. Clin. Genet. 6: Schefer, E. J., S. Lmon-Fv, J. L. Jenner, J. R. McNmr, J. M. Ordovs, C. E. Dvis, J. M. Abolfi, K. Lippel, nd R. I. Levy Lipoprotein[] levels nd risk of coronry hert disese in men. The Lipid Reserch Clinics Coronry Primry Prevention Tril. J. Am. Med. ASSOC. 271: Ridker, P. M., C. H. Hennekens, nd M. J. Stmpfer A prospective study of lipoprotein [] nd the risk of myocrdil infrcti0n.j. Am. Med. Assoc Jurgend, G., nd P. Koltringer Lipoprotein[] in ischemic cerebrovsculr disese: new pproch to the ssessment of risk for stroke. Neurology. 37: Muri, A., T. Miyhr, M. Fujimoto, M. Mtsud, nd M. Kmetm Lp[] lipoprotein s risk fctor for coronry hert disese nd cerebrl infrction. Ath~OSd~OSL. 38: Tyrell, J., T. Cooke, M. Reilly, M. Colgn, D. Moore, D. G. Shnik, C. Bergin, nd J. Feels Lipoprotein[] c 1662 Journl of Lipid Reserch Volume 37,1996

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Blfe Initil hypolbuminemi nd hyperlipidemi persist during chronic peritonel dilysis in children. Ped. Dil. Int. 13: Hffner, S. M.,.S. E. Moss, B. E. K. Klein, nd R. Klein Lck of ssocition between lipoprotein[] concentrtion nd coronry hert disese mortlity in dibetes: the Wisconsin epidemiologic study of dibetic retinopthy. Metbolism. 41: Nielsen, F. S., A. I. Voldsgrd, M. A. Gll, P. Rossing, E. Hommel, P. Andersen, J. Dyerberg, nd H. H. Prving Apolipoprotein [] nd crdiovsculr disese in type 2 (non-insulin dependent) dibetic ptients with nd without dibetic nephropthy. Dibetologi. 36: O'Nel et l. Fctors influencing Lp[]prticle size 1663