Plasma Turnover of HDL ApoC-I, ApoC-III and ApoE in Humans. In Vivo Evidence for a Link Between HDL ApoC-III and ApoA-I Metabolism

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1 JLR Ppers In Press. Published on July 16, 2003 s Mnuscript M JLR200 Plsm Turnover of HDL ApoC-I, ApoC-III nd ApoE in Humns. In Vivo Evidence for Link Between HDL ApoC-III nd ApoA-I Metbolism Jeffrey S. Cohn, Rmi Btl, Michel Trembly, Hélène Jcques, Lyne Veilleux, Cludi Rodriguez, Orvl Mmer ^, nd Jen Dvignon Hyperlipidemi nd Atherosclerosis Reserch Group, nd the ^ McGill University Biomedicl Mss Spectrometry Unit, Montrél, Québec, Cnd. Address correspondence to: Dr. Jeffrey S. Cohn, Hyperlipidemi nd Atherosclerosis Reserch Group, Clinicl Reserch Institute of Montrél, 110 Pine Avenue West, Québec, Cnd, H2W 1R7. Tel: (514) Fx: (514) E-mil: cohnj@ircm.qc.c Supplementry key words: triglyceride, cholesterol, therosclerosis, stble isotope, lipoprotein metbolism Short title: HDL poe, poc-i nd poc-iii kinetics Abbrevitions: ABC-A1, ABC-A1 binding cssette protein; po, polipoprotein; CETP, cholesteryl ester trnsport protein; CHD, coronry hert disese; EL, endothelil lipse; ELISA, enzyme-linked immunosorbent ssy; FPLC, fst protein liquid chromtogrphy; GC-MS, gs chromtogrphy - mss spectrometry; HDL, high-density lipoprotein; HL, heptic lipse; LDL, low-density lipoprotein; LPL, lipoprotein lipse; PLTP, phospholipid trnsfer protein; RT, residence time; SR-B1, scvenger receptor clss B type I; TR, trnsport rte; TRL, triglyceride-rich lipoprotein Copyright 2003 by Lipid Reserch, Inc.

2 Abstrct Numerous fctors re known to ffect the plsm metbolism of high-density lipoproteins (HDL), including lipoprotein receptors, lipid trnsfer proteins, lipolytic enzymes nd HDL polipoproteins (pos). In order to better define the role of HDL pos in determining plsm HDL concentrtions, the im of the present study ws: ) to compre the in vivo rte of plsm turnover of HDL pos (i.e., poa-i, poc-i, poc-iii nd poe), nd b) to investigte to wht extent these metbolic prmeters re relted to plsm HDL levels. We thus studied 16 individuls with HDL cholesterol (HDL-C) levels rnging from mmol/l nd HDL poa-i levels rnging from mg/dl. Plsm kinetics of HDL pos were investigted using primed constnt (12h) infusion of deuterted leucine. Plsm HDL po levels were 41.8 ± 1.5, 9.7 ± 0.5, 4.9 ± 0.5 nd 0.7 ± 0.1 µmol/l for poa-i, poc-i, poc-iii nd poe. Plsm trnsport rtes (TR) were: ± 24.7, ± 12.5, 66.5 ± 9.1, nd 31.4 ± 3.3 nmol.kg -1.dy -1, nd residence times (RTs) were: 5.1 ± 0.4, 3.7 ± 0.3, 3.6 ± 0.3 nd 1.1 ± 0.1 dys, respectively. HDL-C nd poa-i levels were significntly correlted with HDL poa-i RT (r = 0.69 nd r = 0.56), nd were not significntly correlted with HDL poa-i TR. In contrst, HDL poe, poc-iii nd poc-i levels were ll positively relted to their TRs nd not their RTs. HDL poc-iii TR ws postively correlted with levels of HDL poc-iii (r = 0.73, P < 0.01), nd with those of HDL-C nd poa-i (r = 0.54 nd r = 0.53, P < 0.05, resp.). HDL poc-iii TR ws in turn relted to HDL poa-i RT (r = 0.51, P < 0.05). Together, these results provide in vivo evidence for link between the metbolism of HDL poc-iii nd poa-i, nd suggest role for poc-iii in the regultion of plsm HDL levels. Introduction - 2 -

3 Low plsm levels of high-density lipoprotein (HDL) re ssocited with incresed risk of coronry hert disese (CHD) (1,2). A strong therpeutic rtionle therefore exists for incresing plsm HDL levels of ptients t risk of CHD. An ongoing serch for novel therpeutic gents ble to increse plsm HDL levels (3), depends on cler understnding of the mny metbolic fctors which ffect plsm HDL metbolism. These include: ) lipoprotein receptors, e.g., scvenger receptor clss B type I (SR-BI) nd the denosine triphosphte-binding cssette trnsporter (ABCA1) (4,5); b) lipid trnsfer proteins, e.g., cholesteryl ester trnsfer protein (CETP) nd phospholipid trnsfer protein (PLTP) (6); c) lipolytic enzymes, e.g., lipoprotein lipse (LPL), heptic lipse (HL) nd endothelil lipse (EL) (7), nd d) HDL polipoproteins (pos), e.g., poa-i, poa-ii (8). Mny studies hve investigted the physiologicl role of polipoproteins which redily exchnge between plsm lipoproteins (i.e., poc-i, poc-iii nd poe). The mjority of this work hs focussed on the effect of these proteins on triglyceride-rich lipoprotein (TRL) metbolism, however there is mple evidence tht these proteins cn lso directly ffect plsm HDL metbolism (9,10). For exmple, poc-i hs been shown to ctivte lecithin:cholesterol cyltrnsferse (LCAT) (11-13), inhibit heptic lipse (14,15), inhibit phospholipse A 2 (16), nd lso reduce cholesterol ester trnsfer protein ctivity (17,18). ApoC-III nd poe hve both been shown to interct with SR-BI (19,20) nd ABCA1 (21). ApoE cn lso reduce heptic lipse ctivity (22) nd inhibit cubilin-medited HDL endocytosis (23). Direct effects of poc-i, poc-iii nd poe on HDL metbolism re dditionl to their indirect effects, whereby stimultion or inhibition of TRL ctbolism by these proteins could subsequently cuse reciprocl chnge in HDL formtion

4 In order to better define the role of HDL pos in determining plsm HDL concentrtion, the im of the present study ws: ) to compre the in vivo rte of plsm turnover of HDL pos (i.e., poa-i, poc-i, poc-iii nd poe), nd b) to investigte to wht extent these metbolic prmeters re relted to plsm HDL levels. Dt were obtined from 16 individuls who hd previously prticipted in stble isotope-infusion studies focussing on the metbolism of poc-i, poc-iii nd/or poe in TRL (24-26). These subjects hd quite different HDL concentrtions, with three-fold rnge in HDL cholesterol (HDL-C) nd 2-fold rnge in plsm poa-i. Since the intrvenous infusion of deuterted leucine in stble isotope study results in lbelled leucine being incorported into ll plsm proteins, we were ble to retrospectively isolte poa-i from stored blood smples tken from study subjects nd directly mesure poa-i kinetic prmeters for these individuls. This provided unique opportunity to compre plsm mss nd kinetic prmeters for poe, poc-i, poc-iii nd poa-i in HDL

5 Methods Study subjects. Plsm po smples were nlyzed from 16 individuls (15 mles nd 1 femle), who hd prticipted in previous stble-isotope kinetic studies (24-26). They were pprently helthy individuls, ged from 25 to 67 yers with BMI from 23.5 to 30.2 kg/m 2 (Tble I), hving norml or elevted plsm lipid levels. They were not tking medictions known to ffect plsm lipid levels. They hd men (±SE) plsm triglyceride concentrtion of 2.60 ± 0.44 mmol/l, men plsm cholesterol concentrtion of 5.68 ± 0.43 mmol/l, nd n LDL cholesterol concentrtion of 3.68 ± 0.40 mmol/l. Their plsm HDL cholesterol concentrtions rnged from 0.56 to 1.66 mmol/l, nd their plsm poa-i rnged from 89 to 149 mg/dl (Tble I). All subjects gve informed consent to the study protocol, which ws pproved by the Ethics Committee of the Clinicl Reserch Institute of Montrel. Stble isotope infusion. After 12-h overnight fst, subjects were given primed constnt intrvenous infusion of deuterium-lbeled leucine ([D 3 ]L-leucine 98%, Cmbridge Isotope Lbortories, MA), s previously described (27,28). They were injected vi needle ttched to left forerm vein with 10 µmol per kg body weight of [D 3 ]L-leucine, dissolved in physiologicl sline, followed by 12-h constnt infusion (given by peristltic pump) of 10 µmol [D 3 ]L-leucine per kg per h. Subjects remined fsted during the infusion but hd free ccess to drinking wter. Blood smples (20 ml) were collected from n ntecubitl vein of the right rm t regulr intervls (0, 15, 30, nd 45 min, nd 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, nd 12 h) in tubes contining EDTA to finl concentrtion of 0.1%. Plsm ws immeditely seprted by centrifugtion t 3,500 rpm for 15 min t 4 o C. An ntimicrobil gent (sodium zide) nd protese inhibitor (protinin) were dded to plsm smples to give finl concentrtion of 0.02 % nd 1.67 µg/ml, respectively. Lipids, lipoproteins nd polipoproteins. VLDL, IDL + LDL, HDL nd d > 1.21 g/ml - 5 -

6 frction were isolted from 5 ml of plsm by sequentil ultrcentrifugtion in n XL-90 ultrcentrifuge, using 50.4 Ti rotor (Beckmn, 50,000 rpm for 10 h), t densities (d) of 1.006, nd 1.21 g/ml, respectively. All lipoprotein frctions were recovered by tube-slicing nd were djusted to volume of 2.5 ml with physiologicl sline. ApoC-I in HDL ws seprted from other polipoproteins by preprtive electrophoresis using polycrylmide-ure (8M) gels (ph 9.1) (29). ApoA-I, poc-iii nd poe were seprted by preprtive isoelectric focusing (IEF) on 7.5% polycrylmide-ure (8M) gels (ph grdient 4-7) (30). Coumssie blue stining ws used to identify the position of polipoproteins in gels fter electrophoresis. Plsm nd lipoprotein frctions were ssyed for totl (free nd esterified) cholesterol nd triglyceride with COBAS MIRA-S utomted nlyzer (Hoffmn-LRoche) using enzymtic regents. Plsm poa-i concentrtions were mesured by nephelometry on Behring Nephelometer 100 (Behring) using Behring protocol nd regents. Since > 90% of plsm poa-i ws consistently found in the HDL frction fter ultrcentrifugtion (1.063 < d < 1.21 g/ml), plsm poa-i concentrtions were tken to represent HDL poa-i levels. Plsm nd HDL poc-i, poc-iii nd poe concentrtions were mesured with ELISAs developed in our lbortory (26,31,32). Immunopurified polyclonl got nti-humn ntibodies (Biodesign, Kennebunk, ME) were used for both cpture nd detection. Assys were clibrted with stndrd plsms kindly provided by Dr. Petr Alupovic (Oklhom Medicl Reserch Foundtion, Oklhom City). Intrssy nd interssy CVs were between 2% nd 10%. As explined before (24-26), poc-i, poc-iii nd poe recovered in the d > 1.21 g/ml frction (1.9 ± 0.4, 0.5 ± 0.1 nd 14.6 ± 0.9% of totl, resp.) were included in HDL frctions, which were in turn corrected so tht totl polipoprotein recovery fter ultrcentrifugtion ws 100%. Determintion of isotopic enrichment. Apolipoprotein bnds, s well s blnk (nonprotein contining) gel slices were excised from polycrylmide gels. Ech slice ws - 6 -

7 dded to borosilicte smple vil contining 600 µl of 6N HCL, nd n internl stndrd of 250 ng norleucine (Sigm-Aldrich) dissolved in 50 µl double distilled wter. Gel slices were hydrolyzed t 110 o C for 24 h, cooled to -20 o C for 20 min, nd centrifuged t 3,500 rpm for 5 min. Free mino cids in the hydrolyste were seprted from precipitted polycrylmide, purified by ction exchnge chromtogrphy using AG 50 W-X8 resin (BioRd), nd derivtized by tretment with 200 µl of cetyl chloridecidified 1-propnol (1:5 V/V) for 1 h t 100 o C, nd 50 µl of heptflurobutyric nhydride (Supelco) for 20 min t 60 o C (27). Plsm mino cids were lso seprted by ction exchnge chromtogrphy nd derivtized to llow for the determintion of plsm leucine isotopic enrichment. Enrichment of smples with deuterium-lbeled leucine ws mesured by gs chromtogrphy/mss spectrometry (Hewlett-Pckrd, 5988 GC-MS) using negtive chemicl ioniztion nd methne s the modertor gs. Selective ion monitoring t m/z =352 nd 349 (ionic species corresponding to derivtized deuteriumlbeled nd derivtized non-deuterium-lbeled leucine, respectively) ws performed, nd trcer to trcee rtios were derived from isotopic rtios for ech smple. Trcer to trcee rtios were corrected for bckground leucine in gel slices (nd for leucine introduced during the mino cid purifiction nd derivitiztion procedures) by estimting the mount of leucine in processed blnk gel slices in reltion to the norleucine internl stndrd. Bckground leucine represented 10% or less of totl leucine recovered in polipoprotein smples. Kinetic nlysis. Stble isotope enrichment curves for HDL pos were fitted to three comprtment model, s crried out previously (24-26), using SAAM II computer softwre (SAAM II institute, WA). The first comprtment represented the plsm mino cid precursor pool. The second comprtment ws dely comprtment, which ccounted for the synthesis, ssembly nd secretion of polipoprotein. The third comprtment ws the plsm protein comprtment. Plsm leucine enrichment - 7 -

8 (mesured 8 times during the course of the infusion experiment) ws used s mesure of precursor pool enrichment. VLDL pob-100 enrichment t plteu is better estimte of precursor pool enrichment, but since this plteu ws not well-defined in some subjects with elevted VLDL pob-100, plsm leucine ws used for ll individuls. (Since the plsm leucine plteu is usully 10% to 15% higher thn tht of VLDL pob-100, this my hve resulted in 10-15% underestimtion of FCRs for ll polipoproteins). Men (± SE) plsm leucine trcer to trcee rtio ws ± 0.56% (n = 16). Modeling of trcer to trcee rtio dt resulted in the determintion of frctionl trnsport rtes (FTR) (i.e., the frction of HDL po pools being renewed per dy). Residence time (RT) ws clculted s the reciprocl of FTR (1/FTR), nd trnsport rte (TR) ws clculted (in mg.kg -1 dy -1 ) s: where: TR = FTR (pools/dy) X polipoprotein pool size (mg) body weight (kg) pool size = plsm concentrtion (mg/dl) X plsm volume (0.045 liter/kg). Plsm po levels nd HDL po trnsport rtes were expressed in molr units (µmol/l nd nmol/kg.dy) using moleculr weight of 6,613 dltons for poc-i, 8,746 dltons for poc-iii, 34,200 dltons for poe, nd 28,331 dltons for poa-i. Sttisticl nlysis. Sttisticl significnce of differences between men vlues ws ssessed by pired t-tests using SigmStt softwre (Jndel Scientific, CA). T-test results were djusted for multiple comprisons with Bonferroni correction. Person correltion coefficients (r) were clculted to describe the correltion between different kinetic nd mss prmeters

9 Results Chrcteristics of study subjects. Plsm lipid nd po concentrtions of study subjects (n = 16) in the fsted stte, on the dy of the stble-isotope infusion experiment, re shown in Tble I. Subject no. 13 is the femle. Ech vlue represents the verge of 5 mesurements tken t 3-hourly intervls during the infusion. Subjects hd three-fold rnge in HDL cholesterol concentrtion nd 2-fold rnge in plsm poa-i concentrtion. Their men HDL cholesterol concentrtion ws 1.02 ± 0.09 mmol/l nd their men plsm poa-i concentrtion ws ± 4.1 mg/dl (Tble I). Incorportion of lbeled leucine into HDL polipoproteins. Incorportion of deuterted leucine into HDL polipoproteins ws mesured s percentge chnge in trcer to trcee rtio (%ttr) over the 12-hr infusion period (Fig. 1). Rte of chnge in %ttr ws liner for ll 4 polipoproteins. HDL poe %ttr incresed the most rpidly, poa-i %ttr incresed the lest rpidly, nd poc-iii nd poc-i were intermedite, nd were virtully indistinguishble. HDL polipoprotein kinetics. Plsm concentrtion, rte of trnsport nd residence time of HDL polipoproteins re shown in Tble II. Plsm HDL poe concentrtion (in mg/dl) ws significntly lower thn tht of HDL poc-iii, which ws in turn significntly lower thn tht of HDL poc-i, which ws in turn significntly lower thn tht of HDL poa-i (P < 0.01). In molr terms, the concentrtion profile of HDL polipoproteins remined the sme, such tht the concentrtion of HDL poe < HDL poc-iii < HDL poc-i < HDL poa-i. The verge rtio of molecules in the HDL frction ws 1:7:14:60 (i.e., poe:poc-iii:poc-i:poa-i). HDL polipoprotein trnsport rtes were - 9 -

10 determined by computer nlysis (SAAM II) of polipoprotein enrichment curves nd were expressed in units of mg.kg -1.dy -1 nd in units of nmol.kg -1.dy -1. In terms of mg.kg -1.dy -1, poa-i hd the highest rte of trnsport nd HDL poc-iii hd the lowest rte of trnsport, with HDL poe nd poc-i being intermedite. In molr terms however, ll four HDL polipoproteins hd significntly different rtes of trnsport (P < 0.01), nd in mgnitude were ordered similr to HDL molr concentrtions (i.e., poe < poc-iii < poc-i < poa-i). In reltive terms, the rtio between verge molr trnsport rtes ws 1:2:4:12 (i.e., poe:poc-iii:poc-i:poa-i). The verge plsm residence time of poe in HDL ws 1.1 dy, while the verge residence time of HDL poa-i ws 5.1 dys. The verge plsm residence times of HDL poc-iii nd poc-i were very similr (3.6 nd 3.7 dys respectively) which were intermedite between HDL poe nd poa-i. Men frctionl ctbolic rtes (i.e., the reciprocl of residence time) were therefore 0.91, 0.28, 0.27, nd 0.20 pools/dy, for HDL poe, poc-iii, poc-i nd poa-i, respectively. Reltionship between HDL polipoprotein levels nd kinetic prmeters. Simple liner regression nlysis ws crried out to determine which HDL prmeters were sttisticlly correlted (Tble III). As expected, HDL cholesterol nd HDL poa-i concentrtions were significntly correlted (r = 0.52, P < 0.05). A sttisticlly significnt correltion ws lso observed between HDL poc-iii nd HDL cholesterol (r = 0.66, P < 0.01) nd between HDL-poC-III nd HDL poa-i (r = 0.72, P < 0.01). The remining correltion coefficients in Tble III pertin to the reltionship between HDL levels nd HDL kinetic prmeters (left-hnd bottom corner) nd between HDL kinetic prmeters themselves (bottom right). As expected from previous studies, HDL poa-i

11 nd HDL cholesterol levels were significntly correlted with HDL poa-i residence times (r = 0.56 nd r = 0.69), nd were not significntly correlted with HDL poa-i trnsport rtes. This result is shown grphiclly in Fig. 2. In contrst, HDL poe, poc- III nd poc-i levels were ll positively relted to their trnsport rtes nd not their residence times. Positive reltionships between trnsport rtes nd HDL poe, poc-iii nd poc-i levels (r = 0.66, P < 0.01; r = 0.73, P < 0.01 nd r = 0.62, P < 0.05, resp.), re shown in Fig. 2, contrsting to dt for poa-i. Importntly, HDL poc-iii trnsport rtes were postively correlted with both HDL cholesterol nd poa-i levels (r = 0.54 nd r = 0.53, P < 0.05, resp.), nd HDL poc-iii trnsport rtes were in turn relted to HDL poa-i residence times (r = 0.51, P < 0.05)

12 Discussion The present study hs llowed for direct comprison to be mde between the plsm concentrtion nd kinetics of four HDL polipoproteins: poa-i (the principle structurl protein of HDL) nd poc-i, poc-iii nd poe (three polipoproteins which cn trnsfer or exchnge between TRL nd HDL). Our results show tht in terms of mss, there ws pproximtely 2-times more poc-iii thn poe in the HDL frction isolted by ultrcentrifugtion. There ws pproximtely three-times more poc-i thn poe, nd nerly 50-times more poa-i thn poe. Tking into ccount the different moleculr weights of these proteins, the molr rtio in the HDL frction ws 1:7:14:60 (poe:poc-iii:poc-i:poa-i). In obtining these rtios, it is importnt to note tht we hve ssumed tht ny poc-i, poc-iii or poe in the d > 1.21 g/ml frction ws HDL polipoprotein. For poc-i nd poc-iii, this represented less thn 3% of totl plsm, however for poe there ws 14.6 ± 0.9% of totl poe in the bottom frction. It is generlly ccepted tht this represents poe tht is stripped wy from lipoproteins during sequentil ultrcentrigtion (33,34). As we hve proposed previously (25), there re t lest three resons to support the fct tht poe in the d > 1.21 g/ml frction should be regrded s HDL poe: 1) FPLC seprtion of plsm lipoproteins (which voids the high sheer stress of UTC nd therefore does not result in dissocition poe from lipoproteins) provides n HDL poe concentrtion of 2.3 ± 0.3 mg/dl for normolipidemic subjects (NL, n = 12) nd 2.0 ± 0.3 for combined hyperlipidemic subjects (CHL, n = 12) (32). Combining HDL nd d > 1.21 g/ml poe gives similr concentrtions for HDL poe (i.e., 2.9 ± 0.3 mg/dl for NL subjects nd 2.1 ± 0.3 mg/dl for CHL subjects) (25). 2) When poe ws isolted from the d > 1.21 g/ml frction by ffinity chromtogrphy in single untreted ptient, its kinetics were found to resemble

13 tht of HDL poe rther thn VLDL poe (25). 3) Percentge recovery of poe in the bottom frction tends to be lower in hypertriglyceridemic (HTG) versus NL subjects, despite the fct tht the percentge of plsm poe ssocited with TRL is 2-3 fold higher in HTG subjects. ApoA-I cn lso be stripped from HDL by ultrcentrifugtion (35), nd for this reson we hve tken totl plsm poa-i concentrtion to represent poa-i in HDL. Assuming tht there re 2-4 molecules of poa-i in ech HDL prticle, the forementioned dt suggest tht less thn 10% of ll HDL prticles in the circultion contin poe, less thn hlf contin poc-i nd bout two-thirds contin poc-iii. These polipoproteins re not evenly distributed mong poa-i-contining prticles since, for exmple, poe is found in lrger ( nm) prticles, which tend to hve slow pre-bet migrtion on grose gels. ApoC-III on the other hnd, like poa-ii, is found in smller (5-12 nm) α-migrting HDL (36). Some HDL prticles evidently contin more thn one exchngeble polipoprotein, or lterntively more thn one copy of poc-i, poc-iii nd/or poe. In view of this prticle heterogeneity, it is not surprising tht HDL pos hve significntly different rtes of trnsport nd significntly different residence times (Tble II). In molr terms, poe hd the lowest nd poa-i hd the highest TR nd the rtio between TRs ws 1:2:4:12 (poe:poc-iii:poc-i:poa-i). Ech molecule of poe remined in the HDL frction for n verge of one dy, while ech molecule of poa-i remined in HDL for n verge of 5 dys. ApoC-III nd poc-i on the other hnd hd intermedite RTs between 3.5 nd 4 dys. As reviewed by Mrsh et l. (37), 10 previous stble-isotope studies together provided weighted verge of 11.5 ± 2.8 mg.kg -1.dy -1 for HDL poa-i TR nd ± pools/dy for poa-i frctionl ctbolic rte (equivlent to RT of 4.85 dys). These dt compre

14 very well with those obtined for poa-i in the present study (i.e., HDL poa-i TR: 11.0 ± 0.7 mg.kg -1.dy -1 nd HDL poa-i RT: 5.1 ± 0.4 dys). Our dt for HDL poc-i lso compre well with the only other previous poc-i kinetic study by Mlmendier et l. (38). ApoC-I does not contin tyrosine residues nd cn not be lbeled conventionlly with rdioctive iodine. This problem ws overcome by lbelling poc-i with Bolton nd Hunter (BH) regent. More thn 80% of injected 125 I-BH-poC-I ws found ssocited in plsm with HDL (38), nd for 4 NL subjects, poc-i TR ws determined to be 1.8 mg.kg -1.dy -1 nd RT ws 3.2 dys, which compres to 0.9 ± 0.1 mg.kg -1.dy -1 nd 3.7 ± 0.3 dys, resp. in the present study. As fr s poe is concerned, recent study by Hnnuksel et l. (39), reported RTs of 0.60, 0.47 nd 0.38 dys for 125 I-lbeled poe in LpE:A-I:A-II, LpE:A-II or LpE:A-I prticles injected into normolipidemic subjects. These RTs re somewht less thn the 1.1 ± 0.1 dys for HDL poe in this study, which my reflect the fct tht 125 I-lbeled poe cn exchnge or trnsfer more redily from HDL to VLDL thn endogenously lbelled poe. It is certinly striking tht the plsm kinetics of endogenously-lbeled poc-i, poc-iii nd poe re quite different when isolted from HDL rther thn VLDL. Rte of ppernce of deuterted poc-i, poc-iii nd poe is consistently fster in VLDL thn HDL (24-26), suggesting tht in endogenouslbelling experiments crried out in the fsted stte, there is only very slow polipoprotein exchnge or trnsfer between these frctions. Alterntively, there re exchngeble nd non-exchngeble pools of HDL nd VLDL pos, with exchngeble pools being reltively smll compred to the non-exchngeble ones. The presence of exchngeble nd non-exchngeble pools of poc-iii nd poe hs been proposed previously by Bukberg et l. (40) nd Gregg et l. (41,42). An interesting feture of the present dt is tht HDL poa-i concentrtion ws

15 significntly correlted with RT nd not with TR, while levels of HDL poc-i, poc-iii nd poe were significntly correlted with their TRs nd not their RTs (Fig. 2). These dt suggest tht ctbolism rther thn production is the mjor determinnt of HDL poa-i levels, s suggested by previous studies (43-45). In contrst, production rther thn ctbolism is the mjor determinnt of plsm levels of HDL poc-i, poc-iii nd poe. Production, s mesured by rte of trnsport in the present study, could represent: ) direct secretion of poa-i-contining prticles by the liver or intestine contining poc-i, poc-iii nd/or poe; b) synthesis of lipid-poor poc-i, poc-iii nd/or poe, which ssocites with cell membrnes nd is trnsferred to circulting HDL; c) synthesis nd secretion of poc-i, poc-iii nd/or poe on VLDL which is trnsferred or trnsformed into HDL; or d) combintion of the bove. Irrespective of the exct mechnism, it is cler tht the plsm concentrtion of different HDL pos is regulted by different mechnisms nd tht HDL pos re kineticlly s well s dynmiclly different. A finl significnt feture of the present study is the consistent positive correltions tht were observed between HDL poc-iii nd poa-i metbolic prmeters. For the ske of clrity, these sttisticlly significnt reltionships (P < 0.05) re presented digrmticlly in Fig. 3. Firstly, HDL poc-iii levels were found to be positively correlted with those of HDL-C nd HDL poa-i. Secondly, HDL poc-iii trnsport rtes were correlted with HDL-C, HDL poc-iii nd HDL poa-i. And finlly, positive reltionship ws found between HDL poc-iii trnsport rtes nd HDL poa-i residence times, mening tht HDL poc-iii production ws inversely correlted with HDL poa-i ctbolism. Similr reltionships were not observed for HDL poc-i or HDL poe. Plsm levels of HDL were therefore more strongly linked to the metbolism of

16 HDL poc-iii thn to HDL poc-i or HDL poe. This is not to sy tht poc-i nd poe do not hve significnt roles to ply in HDL metbolism, but simply tht the link between poc-iii metbolism nd HDL ws the most evident in vivo. We fvor the view tht this represents n effect of poc-iii on HDL ctbolism nd hence HDL levels, rther thn n effect of HDL levels on poc-iii concentrtion nd production. One possibility is tht incresed production of HDL poc-iii nd/or incresed flux of poc-iii into HDL results in incresed levels of HDL poc-iii nd subsequent inhibition of HDL ctbolism. This is supported by the dt of Luc et l. (46), showing tht in 489 control subjects without CHD, not tking hypolipidemic drugs, poc-iii-lp non-b (mesured by electroimmunssy fter immunoprecipittion of pob-contining lipoproteins) ws significntly correlted with HDL-C nd poa-i concentrtions. ApoE-Lp non-b on the other hnd ws only wekly correlted with HDL-C nd poa-i. Scks et l. hve similrly demonstrted wek but sttisticlly significnt correltion between HDL poc-iii nd HDL cholesterol levels in sub-group of ptients (n = 788) enrolled in the CARE tril (47). Secondly, Stephn et l. (48) hs shown in cebus monkeys tht rte of removl of cholesteryl ester from HDL ws proportionl to the poe to poc-iii rtio of these prticles. Incresed mounts of poc-iii in HDL were thus ssocited with reduced HDL cholesteryl ester removl. And finlly, Le et l. (49) demonstrted in group of 19 subjects tht concentrtions of poc-iii nd poa-i in HDL were significntly correlted (r = 0.73, P < 0.005) nd tht there ws n inverse reltionship between HDL poc-iii concentrtion nd the FCR for poa-i (r = -0.67, P < 0.005). Together, these dt support the concept tht poc-iii my ply n importnt role in regulting HDL ctbolism concept tht deserves further investigtion in isolted tissues nd cultured cells

17 Acknowledgements This study ws mde possible by finncil support (to J.S.C) from the Cndin Institutes of Helth (MT-14684). R.B. received scholrship nd J.S.C. ws lso supported by grnt-in-id, from the Hert nd Stroke Foundtion of Québec. Finncil support from Pfizer Cnd ws lso pprecited. We would lso like to thnk Dr. Mdeleine Roy nd Denise Dubreuil for their help in the clinic, s well s Dr. Hugh Brrett for his ssistnce with the computer nlysis of our kinetic dt

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21 24. Btl, R., M. Trembly, P. H. R. Brrett, H. Jcques, A. Fredenrich, O. Mmer, J. Dvignon, nd J. S. Cohn Plsm kinetics of poc-iii nd poe in normolipidemic nd hypertriglyceridemic subjects. J. Lipid Res. 41: Cohn, J. S., M. Trembly, R. Btl, H. Jcques, L. Veilleux, C. Rodriguez, P. Hugh R. Brrett, D. Debreuil, M. Roy, L. Bernier, O. Mmer, nd J. Dvignon Effect of torvsttin on plsm poe metbolism in ptients with combined hyperlipidemi. J. Lipid Res. 43: Cohn, J. S., M. Trembly, R. Btl, H. Jcques, L. Veilleux, C. Rodriguez, L. Bernier, O. Mmer, nd J. Dvignon Plsm kinetics of VLDL nd HDL poc-i in normolipidemic nd hypertriglyceridemic subjects. J. Lipid Res. 43: Cohn, J. S., D. A. Wgner, S. D. Cohn SD, J. S. Millr, nd E. J. Schefer Mesurement of very low density nd low density lipoprotein polipoprotein (po) B-100 nd high density lipoprotein poa-i production in humn subjects using deuterted leucine: effect of fsting nd feeding. J Clin Invest. 85: Lichtenstein, A. H., J. S. Cohn, D. L. Hchey, J. S. Millr, J. M. Ordovs, nd E. J. Schefer Comprison of deuterted leucine, vline, nd lysine in the mesurement of humn polipoprotein A-I nd B-100 kinetics. J Lipid Res. 31: Kne, J. P A rpid electrophoretic technique for identifiction of subunit species of polipoproteins in serum lipoproteins. Anl. Biochem. 53: Btl, R., M. Trembly, L. Krimbou, O. Mmer, J. Dvignon, J. Genest Jr, nd J. S. Cohn Fmilil HDL deficiency chrcterized by hyperctbolism of mture poa-i but not propoa-i. Arterioscler Thromb Vsc Biol. 18:

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23 38. Mlmendier, C. L., J. F. Lontie, G. A. Grutmn, nd C. Delcroix Metbolism of polipoprotein C-I in normolipoproteinemic humn subjects. Atherosclerosis 62: Hnnuksel, M. L., M. E. Brousseu, S. M. Meyn, H. Nzih, G. Bder, R. D. Shmburek, P. Alupovic, H. B. Brewer Jr In vivo metbolism of polipoprotein E within the HDL subpopultions LpE, LpE:A-I, LpE:A-II nd LpE:A-I:A-II. Atherosclerosis 165: Bukberg, P. R., N-A. Le, H. N. Ginsberg, J. G. Gibson, A. Rubinstein, nd W. V. Brown Evidence for non-equilibrting pools of polipoprotein C-III in plsm lipoproteins. J Lipid Res. 26: Gregg, R. E., L. A. Zech, E. J. Schefer, nd H. B. Brewer Jr Apolipoprotein E metbolism in normolipidemic humn subjects. J Lipid Res. 25: Gregg, R. E., L. A. Zech, E. J. Schefer, D. Strk, D. Wilson, nd H. B. Brewer, Jr Abnorml in vivo metbolism of polipoprotein E4 in humns. J Clin Invest. 78: Schefer, E. J., L. A. Zech, L. L. Jenkins, T. J. Bronzert, E. A. Rublcb, F. T. Lindgren, R. L. Amodt, nd H. B. Brewer Jr Humn polipoprotein A-I nd A-II metbolism. J Lipid Res. 23: Horowitz, B. S., I. J. Goldberg, J. Merb, T. M. Vnni, R. Rmkrishnn, nd H. N. Ginsberg Incresed plsm nd renl clernce of n exchngeble pool of polipoprotein A-I in subjects with low levels of high density lipoprotein cholesterol. J Clin Invest. 91: Brinton, E. A., S. Eisenberg, nd J. L. Breslow Humn HDL cholesterol levels re determined by poa-i frctionl ctbolic rte, which correltes

24 inversely with estimtes of HDL prticle size. Effects of gender, heptic nd lipoprotein lipses, triglyceride nd insulin levels, nd body ft distribution. Arterioscler Thromb. 14: Luc, G., C. Fievet, D. Arveiler, A. E. Evns, J. M. Brd, F. Cmbien, J. C. Fruchrt, nd P. Ducimetiere Apolipoproteins C-III nd E in pob- nd non-pob-contining lipoproteins in two popultions t contrsting risk for myocrdil infrction: the ECTIM study. Etude Cs Temoins sur 'Infrctus du Myocrde. J Lipid Res. 37: Scks, F. M., P. Alupovic, L. A. Moye, T. G. Cole, B. Sussex, M. J. Stmpfer, M. A. Pfeffer, nd E. Brunwld VLDL, polipoproteins B, CIII, nd E, nd risk of recurrent coronry events in the Cholesterol nd Recurrent Events (CARE) tril. Circultion. 102: Stephn, Z. F, J. C. Gibson, nd K. C. Hyes The po E/po CIII molr rtio ffects removl of cholesterol ester from modified humn lipoproteins injected into cebus monkeys. Life Sci. 38: Le, N. A., J. C. Gibson, nd H. N. Ginsberg Independent regultion of plsm polipoprotein C-II nd C-III concentrtions in very low density nd high density lipoproteins: implictions for the regultion of the ctbolism of these lipoproteins. J Lipid Res. 29: Figure Legends Figure 1. Enrichment of poe (closed dimonds), poc-iii (open squres), poc-i (open tringles) nd poa-i (closed circles) in HDL with deuterted leucine. Dt points represent mens ± SEM for 16 subjects. Error brs re too smll to be seen for the poc-iii, poc-i nd poa-i dt points

25 Figure 2. Reltionship between plsm HDL polipoprotein concentrtions nd their rtes of trnsport nd residence times. Dt which were significntly correlted (P < 0.05) hve liner regression lines. ns = not significnt. Figure 3. Digrm showing sttisticlly significnt reltionships (P < 0.05), defined by r vlues, between HDL poc-iii trnsport rtes, HDL poa-i residence times nd plsm concentrtions of HDL poc-iii, HDL cholesterol nd HDL poa-i

26 Tble 1. Subject chrcteristics Subject Age BMI Plsm Plsm LDL HDL ApoA-I Triglyceride Cholesterol Cholesterol Cholesterol yers kg/m 2 mmol/l mmol/l mmol/l mmol/l mg/dl Plsm lipid nd polipoprotein concentrtions for ech fsted subject represent the verge of mesurements for 5 blood smples tken t 3-hourly intervls during the stble-isotope infusion experiment. Subjects re presented in order of incresing plsm poa-i concentrtion.

27 Tble 2. Plsm concentrtion, rte of trnsport nd residence time of HDL polipoproteins HDL ApoE 2.4 ± 0.2 HDL ApoC-III 4.2 ± 0.4 HDL ApoC-I 6.4 ± 0.4 HDL ApoA-I ± 4.1 Concentrtion Rte of Trnsport Residence Time mg/dl µmol/l mg.kg -1.dy -1 nmol.kg -1.dy -1 dys 0.7 ± ± ± ± ± ± ± ± 0.7 b b 31.4 ± ± ± ± ± ± ± ± 0.4 b b Vlues represent mens ± SE. Dt for the four HDL polipoproteins were compred sttisticlly by pired t-test djusted for multiple comprisons by Bonferroni correction. Results in the sme column shring the sme superscript re significntly different ( P < 0.01).

28 Tble 3. Reltionship between plsm HDL concentrtions nd kinetic prmeters HDL Chol. HDL poa-i 0.52 * HDL poe HDL HDL HDL HDL HDL HDL HDL HDL HDL HDL HDL HDL HDL Chol poa-i poe poc-iii poc-i A-I TR A-I RT E TR E RT C-III TR C-III RT C-I TR C-I RT HDL poc-iii 0.66 ** 0.72 ** 0.16 HDL poc-i HDL poa-i TR HDL poa-i RT 0.69 ** 0.56 * * *** HDL poe TR ** HDL poe RT * HDL pociii TR 0.54 * 0.53 * ** * HDL pociii RT ** HDL poci TR * HDL poci RT ** Vlues represent correltion coefficients (r). Significntly correlted: * P < 0.05, ** P < 0.01.

29 Trcer to Trcee Rtio (%) Time After Strt of Infusion (hours) Figure 1

30 5 5 HDL ApoE (mg/dl) r = 0.66, P < r = 0.25, ns HDL ApoE TR (mg/kg.dy) HDL ApoE Residence Time (dys) HDL ApoC-I (mg/dl) HDL ApoC-III (mg/dl) r = 0.73, P < HDL ApoC-III TR (mg/kg.dy) r = 0.62, P < HDL ApoC-I TR (mg/kg.dy) r = -0.03, ns HDL ApoC-III Residence Time (dys) r = -0.06, ns HDL ApoC-I Residence Time (dys) 250 HDL ApoA-I (mg/dl) Figure r = 0.10, ns HDL ApoA-I TR (mg/kg.dy) r = 0.56, P < HDL ApoA-I Residence Time (dys)

31 0.73 HDL ApoC -III TR HDL-CIII HDL-C HDL-AI Figure 3 HDL ApoA -I RT