Conversion of plasma VLDL and IDL precursors into various LDL subpopulations using density gradient ultracentrifugation

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1 Conversion of plsm V nd IDL precursors into vrious subpopultions using density grdient ultrcentrifugtion Crol A. Mrzett, Dvid M. Foster, nd John D. Brunzell Deprtment of Medicine nd Center for Bioengineering, University of Wshington, Settle, WA Abstrct The contribution of very low density lipoproteins (V) nd intermedite density lipoproteins (IDL) to vrious low density.1ipoprotein () subfrctions ws exmined in three norml subjects nd two with fmilil combined hyperlipidemi. Autologous V + IDL (d< g/ml) or V only (d< g/ml; one subject only) were isolted by sequentil ultrcentrifugtion, iodinted, nd injected into ech subject. The ppernce, distribution, nd subsequent disppernce of rdioctivity into density subpopultions ws chrcterized using density grdient ultrcentrifugtion. These techniques help determine the contribution of precursors to vrious subpopultions defined uniquely for ech subject. The results from these studies hve suggested: I) it took up to severl dys of intrvsculr processing of precursor-derived before it resembled the distribution of the stedy-stte plsm protein; 2) plsm V nd IDL precursors contributed rpidly to brod density rnge of ; 3) the rdiolbeled plsm precursors did not lwys contribute to ll density subfrctions within n individul in proportion to their reltive protein mss s determined by density grdient ultrcentrifugtion; 4) with time, the distribution of the precursor-derived becme more buoynt or more dense thn distribution of the protein mss; nd 5) the kinetic chrcteristics of precursor-derived prticles within chnged within reltively nrrow density rnge nd were not lwys relted to the density heterogeneity of ech subject. These studies emphsize the complexities of pob metbolism nd the need to design studies to crefully exmine the production of vrious subpopultions, the kinetic fte nd interconversions mong the subpopultions, nd ultimtely, their reltionship to the development of therosclerosis. - Mrzett, C. A., D. M. Foster, nd J. D. Brunzell. Conversion of plsm V nd IDL precursors into vrious subpopultions using density grdient ultrcentrifugtion. J Lipid Res : Supplementry key words poprotein B very low density lipoprotein low density lipoprotein hve been chrcterized s clss of heterogeneous prticles tht differ in size, density, lipid nd poprotein composition, nd metbolism (1-8). The fundmentl pthwy describing the production of hs trdi- tionlly been thought to involve the production of V by the liver with the subsequent conversion of V to IDL nd then to by series of events involving the hydrolysis nd removl of triglyceride from the core of the prticles (9, 10). More recently, however, lterntive pthwys for production of hve been suggested which involve the direct production of -like prticles from the liver (11-15) nd/or the rpid conversion of smll nscent V or IDL precursors pools to plsm (16-18). While it hs been suggested tht up to 50% of plsm cn be derived from plsm V-independent sources in normolipidemic subjects (17), it is uncler whether certin subfrctions re derived from specific plsm precursors such s V nd IDL while others re derived preferentilly by direct production. Although severl studies hve exmined the contribution of plsm V precursors to different density cuts of (6, 7, 19), there is little informtion vilble on the reltionships between plsm precursors nd vrious plsm subpopultions. The current studies were designed to exmine the conversion of rdiolbeled V nd IDL precursors into vrious, density subpopultions in three normolipidemic individuls nd two subjects with fmilil combined hyperlipidemi. The distribution of precursor-derived rdioctivity ws followed with time mong the density subpopultions unique to ech subject using density grdient ultrcentrifugtion. Abbrevitions: d, density in g/ml; DGUC, density grdient ultrcentrifugtion; EDTA, ethylenediminetetrcetic cid; FCHL, fmilil combined hypercholesterolemi; fx, frction; IDL, intermedite density lipoprotein; -C, low density lipoprotein cholesterol; LTP, lipid trnsfer protein; SDS-PAGGE, sodium dodecyl sulfte-polycrylmide grdient gel electrophoresis; TPC, totl plsm cholesterol; V-C, very low density lipoprotein cholesterol. Present ddress: Pfizer Centrl Reserch, Deprtment of Metbolic Diseses, Estern Point Rod, Groton, CT Journl of Lipid Reserch Volume 31,

2 Subjects MATERIALS AND METHODS Five subjects, three normolipidemic subjects UT, MC, DW; subjects 1, 2, nd 3, respectively, of ref. 20) nd two subjects with fmilil combined hyperlipidemi (FCHL) (subjects WE nd HM; subjects 4, nd 5, respectively, of ref. 20) were studied s outptients of the Clinicl Reserch Center t the University Hospitl of the University of Wshington fter informed ptient consent nd pprovl by the Humn Subjects Review Committee of the University of Wshington. Detiled clinicl prmeters for ech subject hve been published elsewhere (20). Men totl plsm cholesterol, triglyceride, nd pob concentrtions in the control nd FCHL groups were 178 * 50, 59 f 15, 94 f 31, nd 270, 160, 142 mg/dl, respectively. The subjects with FCHL were dignosed ccording to the criteri described previously (20). Subject HM ws tking ethinyl estrdiol (0.05 mg, qd). No other subject ws receiving mediction known to lter lipid metbolism. Ech subject mintined hidher usul diet throughout the study. Potssium iodide (500 mg) ws given orlly three times dy beginning 3 dys before the injection of rdiolbeled lipoproteins nd throughout the study. Preprtion of rdiolbeled lipoproteins A blood smple ws tken from ech subject fter n overnight fst nd plced immeditely into tubes on ice contining 1 mg/ml EDTA, 1 mg/ml NN3, nd 10 U protinin (finl concentrtions). Plsm ws isolted by centrifugtion t 2600 g for 30 min t 4OC. V (d< g/ml) or V + IDL (d< g/ml) nd (1.019 < d < g/ml) were isolted from plsm by sequentil ultrcentrifugtion using 60 Ti rotor (20) Between 3.5 to 5.3 mg of lipoprotein protein ws iodinted with lz5i or I3lI (New Englnd Nucler, Boston, MA) by the method of McFrlne (21) s modified by Bilheimer, Eisenberg, nd Levy (22). Less thn 3% of the rdioctivity in the V or V + IDL preprtions ws free iodine. An verge of % of the totl rdioctivity ws isolted in pob-100 s mesured by SDS- PAGGE nd % ws lipid-extrctble. The rdiolbeled lipoproteins were sterilized using 0.2-pm filter nd checked for pyrogenicity before injection. Design of the studies Ech subject ws injected simultneously with 50 pci of I3'I-V (subject WE) or lz5i-lbeled V + IDL (subjects JT, MC, DW, nd HM) nd 50 pci of lz5i- or I3'I-lbeled fter n overnight fst. The turnover studies hve been described in detil previously (20). Blood smples were obtined 10 min, 3, 6, 9, 24, 34, 48, nd 58 h, nd then dily (8 AM fter n overnight fst) for up to 9 dys fter the injection of rdiolbeled lipopro- teins. Plsm ws isolted from ech blood smple s described erlier nd liquots were tken to determine totl whole plsm rdioctivity. Rdioctivity ws quntitted using Pckrd 5160 gmm counter (Lhun Hills, CA) nd corrections were mde for bckground, spillover, quench, nd decy of ech isotope. Lipoprotein seprtion nd chrcteriztion The density distribution of the rdiolbeled lipoproteins ws determined using density grdient ultrcentrifugtion (DGUC; 20). Briefly, whole plsm ws underlyered into discontinuous slt grdient nd subjected to ultrcentrifugtion in n SW-41 rotor t 41,000 rpm for 24 h t 15OC. For subject HM, V (d< g/ml) ws first isolted from whole plsm by ultrcentrifugtion in 40.3 rotor t 40,000 rpm for 18 h t 10 C nd the d> g/ml frction ws then subjected to DGUC s described. For ech DGUC run, tube contining the sme density solutions but without the lipoprotein smple ws prepred t the sme time. After ultrcentrifugtion, ech smple ws drined nd frctions were collected s described previously (20) The refrctive index (ABBE-3L Refrctometer; Busch nd Lomb, Rochester, NY) ws mesured t 15OC on lternte frctions of the blnk smple. The density for ech frction ws determined using stndrd solutions of known densities nd their refrctive index. The distribution of rdioctivity mong the lipoprotein frctions ws determined by counting ech frction nd correcting for bckground, spillover, quench, nd decy. The verge percentge of recovery of rdioctivity fter DGUC for representtive study ws % (men SD; n = 12). The DGUC profiles tht reflect reltive protein mss or bsorbnce t 280 nm re referred to s protein mss for simplicity. ApoB rdioctivity determintions nd chemicl nlyses After DGUC, specific frctions were pooled bsed on the reltive protein profiles of ech subject (rbitrrily designted s V, IDL, nd l nd B). ApoB rdioctivity ws determined in ech pooled subfrction (defined specificlly for ech subject throughout the text) by isopropnol precipittion (23) nd SDS-polycrylmide grdient gel electrophoresis (SDS-PAGGE) to check for pob-48 contmintion (24). For SDS-PAGGE, the bnds corresponding to ech poprotein were sliced nd counted. In ll studies, less thn 3% of the totl rdioctivity ws isolted in the pob-48 bnd. The plsm disppernce curves for ech individul frction within l nd z were corrected for pob rdioctivity bsed on the percentge of rdioctivity isolted in pob within ech pooled subpopultion. Totl plsm cholesterol nd triglyceride nd lipoprotein cholesterol concentrtions were mesured ccording 976 Journl of Lipid Reserch Volume 31, 1990

3 to the methods outlined by the Lipid Reserch Clinic (25). ApoB mesurements on the pooled lipoprotein subpopultions (V, IDL,,, nd z) were determined using the RIA methods of Albers, Cbn, nd Hzzrd (26). RESULTS The disppernce of V + IDL-derived pob rdioctivity nd its ppernce into the density rnge (s defined by the DGUC profiles) for representtive normolipidemic subject UT) nd FCHL subject (HM) re shown in Fig. 1. In the normolipidemic subject JT, the mximum ppernce of precursor-derived pob rdioctivity into (21.2%) occurred pproximtely 24 h fter injection of the rdiolbled lipoproteins, wheres in FCHL subject HM, this pek occurred pproximtely 48 h fter the injection of the rdiolbeled lipoproteins nd represented 18.0% of the injected pob rdioctivity. For ll subjects, the mximl ppernce of precursor-derived pob rdioctivity into the density rnge vried between 10 nd 48 h fter the injection of the % 8 JT V+IDL Fig. 1. Disppernce of rdiolbeled V + IDL pob rdioctivity fmm plsm nd its ppernce into s defined by DGUC; A, V + IDL; 0,. The symbols represent the observed dt, the lies re drwn by hnd. JT, normolipidemic; HM, FCHL. rdiolbeled lipoproteins nd ws positively correlted to totl plsm cholesterol (TPC) concentrtions [Y = 0.85; time of pek = 0.008(TPC) ; P < 0.007], plsm TG concentrtions [r = 0.99; time of pek = (E) ; P = , plsm pob concentrtions [r = 0.84; time of pek = 0.014(poB)-0.427; P = 0.071, V cholesterol (V-C) concentrtions [Y = 0.94; time of pek = 0.088(V-C) ; P < 0.021, nd cholesterol (-C) concentrtions [Y = 0.63; time of pek = O.OOG(-C) ; P = Since ech dt point long the plsm disppernce nd ppernce curves ctully represents the sum of the rdioctivity distributed mong ll the lipoprotein subpopultions, the distribution of the precursor-derived pob rdioctivity mong the vrious products ws exmined in more detil using DGUC. For ll subjects, the distribution of the rdioctivity t the time in which the mximl ppernce of precursor-derived pob rdioctivity ws isolted within the density rnge ws never superimposble with the distribution of the protein mss. As shown in normolipidemic subject JT, t 24 h (mximum pob rdioctivity; see Fig. l), the pek density of the rdiolbeled lipoproteins distributed primrily in more buoynt region (1.034 g/ml) compred to the plsm protein mss (1.041 g/ml Fig. 2, 1 dy). Similrly, in FCHL subject HM, t 48 h (mximum pob rdioctivity; see Fig. 1) the pek density of the rdiolbeled lipoproteins ws g/ml compred to the pek density of the mss which ws g/ml (Fig. 3, 2 dys). With time, vrible mounts of precursor-derived pob rdioctivity were isolted within the density rnge defined uniquely by the DGUC profiles for ech subject (12-38%; clculted s the mximum pob rdioctivity isolted within l plus p fter seprtion by DGUC divided by the injected dose). With time, in the normolipidemic subjects the precursor-derived rdioctivity distributed mong the vrious subpopultions nd resembled the protein distribution of the plsm. As shown for normolipidemic subject JT, 3 dys fter the injection of rdiolbeled lipoproteins, the distribution of the rdiolbled lipoproteins resembled the distribution of the plsm protein mss (Fig. 2, 3 dys). However, with time in this subject, the pek density of the rdiolbeled lipoproteins becme more dense (1.047 g/ml) compred to the density of the plsm ( g/ml; Fig. 2, 6 dys). In contrst, in both FCHL subjects the distribution of the precursor-derived rdioctivity never resembled the distribution of the protein mss. As illustrted in Fig. 3, with time the distribution of the precursor-derived rdioctivity remined in more buoynt region (1.039 g/ml) compred to the protein mss (1.043 g/ml) even 7 dys fter the injection of the rdiolbeled lipoproteins (Fig. 3, lower pnel). Mrze6t, Fostq nd Bruntell Conversion of plsm V nd IDL to subpopultions 977

4 4 i x Y >.r(.r( Y 2 0 s E d cd Y 0 c) ru 0 8.r( Y 1 e.r( k v1 s * 20- JT Frction ## Fig. 2. Density grdient ultrcentrifugtion profiles of the distribution of reltive protein mss (-) nd V + IDL-derived totl rdioctivity (A) mong pob-contining lipoproteins isolted from normolipidemic subject JT 1, 3, nd 6 dys fter the injection of rdiolbeled lipoproteins. sor-derived rdioctivity into occurred rpidly in both the buoynt (frctions 14-16; < d < g/ml) nd dense (frctions 17-21; 1.036<d< g/ml) subpopultions. However, pproximtely 10 h fter the injection of the rdiolbeled lipoproteins, rdioctivity within the l subpopultion disppered t rte similr to tht seen in the IDL subpopultion. In contrst, rdioctivity within the, subpopultion continued to increse until bout 1.5 dys nd then disppered t much slower rte compred to l (Fig. 4, JT). In FCHL subject HM, the generl metbolic chrcteristics of the V nd IDL subpopultions were similr to the norml subjects (Fig 4, lower pnel). Tht dys r 4 dys AA/ A-A-A-A HM To exmine the metbolic behvior of vrious lipoproteins in more detil, frctions within specific density rnges (rbitrrily chosen nd designted s V, IDL, 1, nd 2 bsed on the density grdient profiles unique to ech subject) were pooled nd pob rdioctivity ws determined for ech subpopultion s described previously. The disppernce of rdiolbeled pob from V in normolipidemic subject JT (frctions 1-8 d < g/ml; Fig. 4, upper pnel), decresed rpidly within the first dy nd then disppered t slower rte. A prominent dely in the disppernce of rdioctivity ws seen in the IDL subpopultion (frctions 9-13; < d < g/ml). The ppernce of precur- Frction ## Fig. 3. Density grdient ultrcentrifugtion proliles of the distribution of reltive protein mss (-) nd V + IDL-derived totl rdioctivity (A) mong pb contining lipoproteins isolted from FCHL subject HM 2, 4, nd 7 dys fter the injection of rdiolbeled lipoproteins. 978 Journl of Lipid Reserch Volume 31, 1990

5 8 b w JT HM the reltionships between the generl kinetic chrcteristic nd density heterogeneity of ech subject s subpopultions, the ppernce nd disppernce of rdioctivity with time ws exmined in ech frction within l nd 2 s defined by DGUC. Individul frctions within the buoynt nd dense subpopultions hve been corrected for pob rdioctivity determined for ech pooled subpopultion s described erlier. Within the, subpopultion in normolipidemic subject JT, the increse in rdioctivity into frctions 14, 15, nd 16 ws similr nd rpid; however, the pek of the rdiolbel occurred first in the most buoynt frction (fx 14), followed in order by frctions 15 nd 16 (Fig. 5, I). Although the rte of removl fter the initil increse of rdioctivity into ech frction ws reltively rpid, the finl rte of disppernce slowed progressively with in- JT 1.o IDL Fig. 4. The disppernce nd ppernce of pob rdioctivity from V (A), IDL (O),, (o), nd 2 (0) s defined by the DGUC profiles. The symbols represent the observed dt nd the lines re drwn by hnd. JT, normolipidemic; HM, FCHL. is, there ws n initil rpid disppernce of rdioctivity from V (d < g/ml) followed by slower rte of disppernce nd n initil increse of rdioctivity into IDL (frctions 1-8; d< g/ml) followed by rpid nd then slower disppernce from this subpopultion. As in the normolipidemic subject, the rte of ppernce of precursor-derived rdioctivity incresed similrly into buoynt nd dense, however unlike JT, the rte of disppernce from l (frctions 9-13; <d < g/ml) nd :, (frctions 14-19; 1.042<d<1.069 g/ml) fter 2 dys ws nerly identicl (Fig. 4, HM). Similr rtes of disppernce in both subpopultions were lso seen in normolipidemic subjects MC nd DW wheres the disppernce of rdioctivity from l nd P from FCHL subject WE ws more similr to subject JT (dt not shown). Although dividing into buoynt nd dense subpopultions ws bsed on the DGUC protein profiles unique to ech individul, it ws cler tht the density heterogeneity in some of the subjects ws more complex (see Fig. 1 of ref. 20). To obtin dditionl informtion on 1.u Q 1.0 r.n rr j -----w- 4 1x11 fx19 fx21,.,., Fig. 5. The ppernce nd disppernce of V + IDL-derived pob rdioctivity with time in ech frction isolted within the density rnge fter seprtion by DGUC in, (upper pnel) nd vl2 (lower pnel) in normolipidemic subject JT Frction numbers comcide wth thme given in Fig. 2 nd re numbered from buoynt to dense. The symbols represent the okrved dt; the lines re drwn by hnd. Ech symbol represents different frction (fx) s lbeled. Mrzett, Foster, nd Brunzell Conversion of plsm V nd IDL to subpopultions 979

6 cresing density. The initil rte of increse of rdioctivity within frctions within the 2 subpopultion occurred primrily in the more buoynt frctions. Rdioctivity disppered most rpidly from the more buoynt 2 frctions (fx 17 nd 18; < d < g/ml) nd then slowed considerbly in the denser frctions 19 through 21 (1.045 <d< g/ml; Fig. 5, 2). In FCHL subject HM, the initil increse in rdioctivity ws similr nd rpid in frctions 9-13 (1.027 <d< g/ml) within the l subpopultion with the pek of rdioctivity pproximtely 1.5 dys fter the injection of the rdiolbeled lipoproteins (Fig. 6, l). There ws similr rpid rise in rdioctivity within frction 13 (1.042 <d< g/ml) of the l subpopultion but the pek occurred slightly lter (2.5 dys). The disppernce of rdiolbeled prticles from the l subpopultion ws fster in frctions 9 nd IO nd slowed progressively in frctions Within the 2 subpopultion, the pek in rdioctivity occurred dys fter the injection of the iodinted V + IDL (Fig. 6, 2). In contrst to JT, however, the disppernce of rdioctivity ws very similr in ll frctions within the 2 subpopultion nd ws virtully identicl to the rte of clernce of rdioctivity from frction 13 within the l subpopultion. DISCUSSION The current studies were designed to exmine the conversion of plsm precursors to the vrious subpopultions unique to ech individul using density grdient ultrcentrifugtion. Although vrious DGUC or flottion methods hve been used to describe heterogeneity in more detil, only recently hve these methods been used to study the metbolic behvior of lipoproteins (6, 7, 16, 20, 27, 28). DGUC provides some dvntges over sequentil ultrcentrifugtion such s decresed ultrcentrifugtion time, the chrcteriztion of density heterogeneity unique to ech subject, nd greter detil in describing the reltionships between physicl nd kinetic vribility s well s mong precursors nd vrious products (16, 20). In the current studies, the generl density chrcteristics vried mong the subjects with pek densities rnging from g/ml to g/ml In ddition, vrious density subpopultions within n individul were evident in four out of five of the subjects (20). Severl importnt chrcteristics of pob metbolism hve been suggested from the studies presented here. First, it took severl hours to dys fter the mximl conversion of precursor into product for the precursor-derived prticles to resemble plsm (Fig. 1, JT nd Fig. 2). Second, rdiolbeled precursors contributed rpidly to brod density rnge of products q c) 5 0.1, r; 1.0 % u 0 Q) '7 E._ HM u, 0 Y 7 E b) " fx 14 fx 15 l'x 16 fx 17 fx 18 \b- fx 19 l I.,. l.. I I., 1 Fig. 6. The ppernce nd disppernce of V + IDL-derived pob rdioctivity with time in ech frction isolted within the density rnge fter seprtion by DGUC in, (upper pnel) nd 2 (lower pnel) in FCHL subject HM. Frction numbers coincide with those given in Fig. 3 nd re numbered from buoynt to dense. The symbols represent the observed dt, the lines re drwn by hnd. Ech symbol represents different frction (fx) s lbeled. (Le., l nd,; Fig. 4); Third, in two out of the five subjects (HM nd WE), the precursors did not contribute uniformly to ll plsm density subpopultions (Fig. 3). Fourth, with time the distribution of the precursor-derived chnged in reltionship to the individul's plsm protein mss in three out of five subjects UT, MC, nd DW, Fig. 2). And finlly, the kinetic chrcteristics of prticles within ppered to chnge within nrrow density rnge nd were not necessrily relted to the physicl density heterogeneity of ech subject (Fig. 5,,; Fig. 6, 1). Trditionlly, the reltionship between V precursors nd products hs been described by following 980 Journl of Lipid Reserch Volume 31, 1990

7 the disppernce of rdioctivity from V (d < g/ml) nd its subsequent ppernce in (1.019 < d < g/ml) using sequentil ultrcentrifugtion (reviewed in 29). However, the dt obtined using these conventionl methods represents the sum of the rdioctivity distributed mong ll the potentil products (subpopultions). For ech subject in the current studies, the ppernce of precursor-derived rdioctivity s well s the distribution of these rdiolbeled lipoproteins mong subpopultions ws exmined t ech time point using DGUC. Using these techniques, the precursor-derived prticles were more buoynt nd non-superimposble with the protein mss t the time point in which the mximum mount of precursor-derived rdioctivity ws isolted within the density rnge defined uniquely for ech subject (Fig. l vs Figs. 2 nd 3). Although, by definition, ll the rdioctivity isolted within the density rnge represents low density lipoproteins, these studies suggest tht it tkes up to severl dys for the precursor-derived prticles to resemble the plsm density chrcteristics unique to ech subject. These observtions my hve importnt implictions concerning the contribution of plsm precursors to vrious products. Tht is, the contribution of V nd IDL to the mjor dense subpopultion my be fr less thn predicted from the trditionl plsm V precursor- product curves. Detiled quntittive nlyses will help in understnding these reltionships nd re currently underwy. It is of interest to note tht the rte of conversion of V-IDL precursors to products ws sttisticlly correlted to totl plsm cholesterol, V cholesterol, plsm TG, nd pob concentrtions, but not cholesterol concentrtions. In ddition, no sttisticlly significnt correltions were found between these vribles nd the mount of rdiolbeled precursor pob tht ws converted to pob (dt not shown). Although the biologicl significnce of these observtions is not known, it gin reflects the complexity of pob metbolism nd the need to hve better understnding of the reltionships between V nd production. Conceptully, it is thought tht plsm rises from the delipidtion of V to IDL nd then to, presumbly, buoynt to dense. This conversion ppers to involve severl processes including intrvsculr enzymes such s lipoprotein lipse nd heptic lipse which hydrolyze the core triglycerides of the precursor prticles nd the loss of surfce constituents (PL, FC, nd poproteins) which re trnsferred to high density lipoproteins (reviewed in 30). However, the results from the current studies suggest tht plsm precursors contributed rpidly to brod density rnge of. As shown in Fig. 4, the initil ppernce of precursor-derived rdioctivity in l nd 2 ws pproximtely prllel. Tht is, the initil conversion of V + IDL to buoynt (1.026 <d< g/ml; density rnges for both subject s l combined) nd dense (1.036<d < g/ml; density rnges for both subjects 2 combined) subpopultions ws similr. Recently, Teng et l. (6) hve shown the rpid conversion of rdiolbeled plsm V into visully seprted light (men density g/ml) nd hevy (men density g/ml) in normolipidemic subjects, but not in fmilil hypercholesterolemic (FH) subjects. In ddition, Huff nd Telford (31) hve published similr observtions in miniture pigs. It is uncler whether specific subpopultions re derived from certin precursors. For exmple, buoynt subpopultions might be derived preferentilly from specific precursors such s certin V precursors, while the dense subpopultions might be derived preferentilly from IDL precursors. This could help explin the rpid conversion of precursors into buoynt s well s dense subpopultions. This hypothesis nd others cn be tested using complex comprtmentl modeling; however, studies designed to test the preferentil conversion of certin precursors to specific products will need to be done to ddress this possibility directly. In two out of the five subjects (FCHL subjects WE nd HM), the plsm precursors did not pper to contribute equivlently to ll subpopultions. At no time throughout the time course of ech study ws the distribution of the precursor-derived superimposble with the distribution of the plsm protein (Fig. 3, lst pnel). Although it is possible tht, with dditionl time, the precursor-derived prticles were metbolized to the dense subpopultions, the overll contribution of the precursors to these subpopultions would hve been miniml. Tht is, less thn 6% of the injected dose of precursor lipoprotein pob remined in plsm t the end of these studies of which 2.8% nd 1.7% (WE nd HM, respectively) ws isolted within the dense 2 frction. In the three normolipidemic subjects, in t lest one point throughout ech study, the distribution of the precursor-derived resembled the distribution of the plsm protein (Fig. 2, middle pnel). These observtions support the recent work of Teng et l. (6) in which the conversion of V to IDL to buoynt to dense ws seen in normolipidemic nd hyperpobetlipoproteinemic subjects, but not in FH subjects. Although the limited number of study subjects presented here limits our bility to mke generl sttements on the potentil differences in the conversion of plsm precursors to specific products between normolipidemic nd FCHL subjects, it is interesting to speculte on the possibility tht the smll dense chrcteristic of FCHL subjects my be derived from plsm V-independent sources in t lest subset of these ptients. Additionl metbolic heterogeneity within precursorderived ws observed in the normolipidemic subjects. As described bove, t one point in time the Mrzett, Fostet; nd Brunzell Conversion of plsm V nd IDL to subpopultions 981

8 distribution of the precursor-derived resembled the distribution of the plsm protein in the normolipidemic subjects (3 dys for JT, 1 dy for MC, nd 2 dys for DW). However, with time the distribution of the precursor-derived chnged compred to the stedystte distribution of the plsm protein mss. In subject JT, the rdiolbeled incresed in density with time (Fig. 2, lower pnel) while in subjects MC nd DW, the rdiolbeled isolted in buoynt region of the density rnge (dt not shown). Chnges with time in the distribution of rdiolbeled light nd hevy (1.025 < d < nd < d < g/ml, respectively) hve been seen in preliminry studies done in mildly hypertriglyceridemic subjects by Beltz, Young, nd Witztum (27). Similr observtions hve been mde by Luc nd Chpmn (28) in guine pig. It is uncler wht influences the metbolic behvior of these prticles lthough their interctions with intrvsculr enzymes must be importnt. Very few studies hve exmined the substrte specificity of different subpopultions to vrious plsm enzymes. In our lb, preliminry studies hve suggested tht, in some subjects, buoynt subpopultions or metbolic pools within this region my be preferentil cceptors for HDL-derived cholesteryl esters (C. A. Mrzett, T. J. Meyers, nd J. J. Albers, unpublished results). Studies exmining substrte specificity of the key lipoprotein intrvsculr enzymes will be importnt in our understnding of the metbolic heterogeneity within. In the current studies, it ws not possible to obtin pob specific ctivity for ech frction isolted within plsm. Tht is, the percentge of the totl rdioctivity ssocited with pob ws determined for the pooled l nd, subpopultions only. Then, the percentge of pob rdioctivity in l ws used to correct ll the l frctions to pob rdioctivity nd, likewise, the percentge of pob rdioctivity found in B ws used to correct ll the Z frctions. These clcultions could potentilly led to slight vritions in the pob rdioctivity curves since lrger more buoynt re more likely to contin more poe thn the smller more dense (4, 5). However, even if the pob corrections re slightly under- or over-estimted for ny given frction, this would ffect the overll mgnitude of ech curve rther thn chnge the kinetic chrcteristic of the curve, ssuming the poprotein compositions styed constnt throughout ech study. Since density nd kinetic heterogeneity were chrcterized in detil in ech subject, it ws possible to exmine potentil reltionships between the physicl nd metbolic chrcteristics. As illustrted in normolipidemic subject JT, distinct differences in the metbolic behvior of the individul frctions occurred within nrrow density rnge (Fig. 5; frctions 18 nd 19). This observtion ws consistent with the increse in pek den- sity with time in the precursor-derived compred to the stedy-stte protein mss (Fig. 2, lst pnel). These chnges, however, could not be predicted bsed on the physicl chrcteristics of this subject s plsm which ws reltively homogeneous compred to the other subjects. In FCHL subject HM, noticeble lg in the mximum conversion of precursor-derived rdioctivity into ws seen within nrrow density rnge (Fig. 6; pek conversion 1.5 to 2 dys for fxs 9-12 vs 2.5 dys for fx 13). This frction corresponded to the inflection point between the shoulder of the buoynt portion of the DGUC profile (l) nd the more dense mjor pek (,; Fig. 3). This reltionship between discrete chnges in the kinetic behvior of certin frctions nd chnges in the distribution of the protein mss ws lso seen in FCHL subject WE, but no obvious reltionships were seen between the physicl nd kinetic heterogeneity of in the other normolipidemic subjects DW nd MC (dt not shown). Unpredictble differences in the metbolic behvior of specific rdiolbeled density frctions nd the vrious density subpopultions chrcterized by DGUC were lso seen in these sme subjects when injected with rdiolbeled plsm (20). Although chnges in the chemicl compositions of plsm when subdivided into different density rnges pper to chnge grdully nd linerly (2), subtle chnges in the poprotein composition could help ccount for chnges in the kinetic behvior of specific frctions. As shown previously by Gibson et l. (4), lrge buoynt hve more poe ssocited with them thn do smller, more dense. Since poe is n importnt lignd for receptor recognition, it is possible tht the presence of poe in some of these frctions would influence its metbolic fte. Ultimtely, it will be very informtive to be ble to nlyze the lipid nd poprotein composition of ech frction isolted within ech subject s. In summry, the conversion of plsm V nd IDL into vrious subpopultions ws nlyzed using density grdient ultrcentrifugtion in five subjects. The observtions mde using this pproch suggested: I) it my tke severl dys of intrvsculr processing of precursor-derived to resemble the distribution of plsm protein; 2) V nd IDL precursors contributed rpidly to brod density rnge of products; 3) the rdiolbeled plsm precursors did not lwys contribute uniformly to ll subpopultions; 4) with time, the distribution of the precursor-derived chnged in reltionship to the distribution of the protein mss in some subjects; nd 5) the kinetic chrcteristics of prticles within chnged within reltively nrrow density rnge nd were not lwys correlted to the density heterogeneity defined uniquely for ech subject. It is cler from the current studies tht metbolism is very complex. Although density grdient ultrcentrifugtion is just one methodologicl tool 982 Journl of Lipid Reserch Volume 31, 1990

9 in which more detiled observtions on the reltionships between the physicl nd kinetic heterogeneity of cn be mde, other methods such s gel filtrtion (16) nd nondenturing grdient gel electrophoresis (32). Combintions of these methods will help further our knowledge of the production nd subsequent metbolism of vrious subpopultions. HB The uthors would like to thnk the Clinicl Reserch Center stff; John Hoknson nd Jnet Adolphson for their technicl expertise; nd the study subjects for their coopertion. This work ws supported by NIH grnts HL-30086, RR-02176, nd RR-36. C. A. M. ws supported by n Institutionl Ntionl Reserch Service Awrd (HL-07312). Mnuscript receiued 22June 1989, in reuised form 20 October 1989, nd in re-reuised form 26 Jnurv REFERENCES 1. Fisher, W. R., M. F. Hmmond, nd G. L. Wrmke Mesurements of the moleculr weight vribility of plsm low density lipoproteinemi. Demonstrtion of mcromoleculr-heterogeneity. Biochkby. 11: Shen, M. M. S., R. M. Kruss, F. T. Lindgren, nd T. M. Forte Heterogeneity of serum low density lipoproteins in norml subjects. J Lipid Res Kruss, R. M., nd D. J. Burke Identifiction of multiple subclsses of plsm low density lipoproteins in norml humns.j LipidRes. 23: Gibson, J. C., A. Rubenstein, P. R. Bukberg, nd W. V. Brown Apolipoprotein E-enriched lipoprotein subclsses in normolipidemic subjects. J. Lipid Res. 24: Lee, D. M., nd P. Alupovic Apolipoprotein B, C-111, nd E in two mjor subpopultions of low-density lipoproteins. Biochim Bwptys. Act 879: Teng, B., A. D. Snidermn, A. K. Soutr, ndg. R. Thompson Metbolic bsis of hyperpobetlipoproteinemi. Turnover of polipoprotein B in low density lipoprotein nd its precursors nd subfrctions compred with norml nd fmilil hypercholesterolemi. J Clin. Invest. 77: Veg, G. L., nd S. M. Grundy Kinetic heterogeneity of low density lipoproteins in primry hypertriglyceridemi. ArteriFclemrir. 6: Foster, D. M., A. Chit, J. J. Albers, R. A. Filor, C. Hrris, nd J. D. Brunzell Evidence for kinetic heterogeneity mong humn low density lipoproteins. Metbolism. 35: Bermn, M., M. Hll, R. I. Levy, S. Eisenberg, D. W. Bilheimer, R. Phir, nd H. Goebel Metbolism of pob nd poc lipoproteins in mn: kinetic studies in norml nd hyperlipoproteinemic subjects. J. Lipid Res. 19: Zech, L. A., S. M. Grundy, D. Steinberg, nd M. Bermn Kinetic model for production nd metbolism of very low density lipoprotein triglycerides. Evidence for slow production pthwy nd results for normolipidemic subjects. J Clin Invest. 63: Johnson, F. L., R. W. St. Clir, nd L. L. Rudel Studies on the production of low density lipoproteins by perfused livers from Africn green monkeys. Effects of dietry cholesterol. J Clin. Invest. 72: Johnson, E L., R. W. St. Clir, ndl. L. Rudel Effects of the degree of sturtion of dietry ft on the heptic production of lipoproteins in the Africn green monkey. J Lipid Res. 26: Johnson, E L., L. L. Swift, nd L. L. Rudel Nscent lipoproteins from recirculting nd nonrecirculting liver perfusions nd from the heptic Golgi pprtus of Africn green monkeys. J Lipid Rtx 28: Jones, L. A., T. Termoto, D. J. Junh, R. B. Goldberg, A. H. Rubenstein, nd G. S. Getz Chrcteriztion of lipoprotein produced by the perfused rhesus monkey liver. J. Lipid Res. 25: Termoto, T., H. Kto, Y. Hshimoto, M. Kinoshit, T. Wtnbe, H. Ok, nd c. Nito Effect of dietry cholesterol on production of lipoproteins nd polipoproteins by perfused livers from Jpnese monkeys (MuccJLrct). Eut: J. C6in. Invest. 17: Mrzett, C. A., E L. Johnson, L. A. Zech, D. M. Foster, nd L. L. Rudel Metbolic behvior of heptic V nd plsm pob-100 in Africn green monkeys. J. Lipid Res. 30: Beltz, W. E, Y. A. Kesniemi, B. V. Howrd, nd S. M. Grundy Development of n integrted model for nlysis of the kinetics of polipoprotein B in plsm very low density lipoproteins, intermedite density lipoproteins, nd low density lipoproteins. J. Clin. Invest Shephrd, J., nd C. J. Pckrd Metbolic heterogeneity in very low-density lipoproteins. Am. HertJ 113: Fisher, W. R., L. A. Zech, P. Brdlye, G. Wrmke, nd M. Bermn Metbolism of polipoprotein B in subjects with hypertriglyceridemi nd polydisperse. J Lipid& 21: Mrzett, C. A., D. M. Foster, nd J. D. Brunzell Reltionships between density nd kinetic heterogeneity in subjects with normolipidemi nd fmilil combined hyperlipidemi using density grdient ultrcentrifugtion. J Lipid Res. 30: McFrlne, A. S Efficient-trce lbeling of proteins with iodine. Nture (London). 182: Bilheimer, D. W., S. Eisenberg, nd R. I. Levy The metbolism of very low density lipoprotein proteins. Preliminry in vitro nd in vivo observtions. Bwchim. Biopt& Act Ymd, N., nd R. J. Hvel Mesurement of polipoprotein B rdioctivity in whole plsm by precipittion with isopropnol. J Lipid Res. 27: Mrzett, C. A., nd L. L. Rudel A species comprison of low density lipoprotein heterogeneity in nonhumn primtes fed therogenic diets. J Lipid Res. 27: Lipid Reserch Clinics Progrm Mnul of Lbortory Opertions, Vol. I: Lipid nd Lipoprotein Anlysis. NIH, Publiction # Albers, J. J., V. G. Cbn, nd W. R. Hzzrd Immuno-ssy of humn plsm polipoprotein B. Metbolism. 24: Beltz, W. F., S. G. Young, nd J. L. Witztum Heterogeneity in low density lipoprotein metbolism. In Proceedings of the Workshop on Lipoprotein Heterogeneity. K. Lippel, editor. NIH Publiction # Luc, G., nd M. J. Chpmn Guine pig low density lipoproteins: structurl nd metbolic heterogeneity. J Lipid Res Kesniemi, Y. A., G. L. Veg, nd S. M. Grundy Kinetics of polipoprotein B in norml nd hyperlipidemic mn. Mrzett, Fostel; nd Brunzell Conversion of plsm V nd IDL to subpopultions 983

10 Review of current dt. In Lipoprotein Kinetics nd Model- cholestyrmine in miniture pigs: effects on composition ing. M. Bermn, S. M. Grundy, nd B. V. Howrd, editors. nd synthesis of subfrctions. Metbolin Acdemic Press, Sn Frncisco, CA Musliner, T. A,, K. M. McVicker, J. E Iosef, nd R. M. 30. Gotto, A. M., H. J. Pownll, nd R. J. Hvel Introduc- Kruss Metbolism of humn intermedite nd very 31. tion to the plsm lipoproteins. Mcthodr Enzymol. 128: low density lipoprotein subfrctions from norml nd Huff, M. W., nd D. E. Telford Regultion of low den- dyslipoproteinemic plsm. In vivo studies in the rt. A~tmclesity lipoprotein poprotein B metbolism by lovsttin nd ~xdslf. 7: Journl of Lipid Reserch Volume 31, 1990