Michael Lefevre,' Charles H. Sloop, and Paul S. Roheim. Animals, diet and peripheral lymph collection

Transcription

1 Chrcteriztion of dog prenodl peripherl lymph lipoproteins. Evidence for the peripherl formtion of lipoprotein-unssocited poa-l with slow pre-p electrophoretic mobility Michel Lefevre,' Chrles H. Sloop, nd Pul S. Roheim Deprtment of Physiology, Louisin Stte University Medicl Center, 1542 Tulne Avenue, New Orlens, LA Abstrct Dog plsm nd prenodl peripherl lymph poa-i distribution ws exmined by nondenturing grdient gel electrophoresis-immunoblot nlysis. In control dogs, plsm poa-i could be loclized to two distinct popultions of prticles with modl dimeters of 8.4 nm nd 1.4 nm. The smller sized popultion ccounted for over 5% of plsm poa-i. Peripherl lymph poa-i distribution ws significntly different. The percentge of poa-i loclized to the 1.4 nm popultion ws reduced by 4% nd the modl dimeter of the smller HDL poa-i popultion ws significntly decresed by.1 nm. Additionlly, peripherl lymph poa-i could be loclized to prticles smller thn lbumin (lipoprotein-unssocited poa-i). The presence of lipoprotein-unssocited poa-i prticles ws confirmed by gel filtrtion chromtogrphy. Immunoblots of column frctions subjected to grose electrophoresis reveled tht these prticles hd slow pre-bet electrophoretic mobility. In dogs fed n therogenic diet, lipoprotein-unssocited poa-i prticles with slow pre-bet electrophoretic mobility could be found in both plsm nd peripherl lymph. With incresing degree of hypercholesterolemi, the reltive mount of plsm lipoprotein-unssocited poa-i tended to increse. In peripherl lymph, n incresing degree of hypercholesterolemi ws ssocited with decrese in the reltive mount of lipoprotein-unssocited poa-i. Insted, popultion of lrge poa-i prticles (11-25 nm) becme incresingly prominent. - Lefevre, M., C. H. Sloop, nd P. S. Roheim. Chrcteriztion of dog prenodl peripherl lymph lipoproteins. Evidence for the peripherl formtion of lipoprotein-unssocited poa-i with slow pre-8 electrophoretic mobility. J. Lipid Res : Supplementry key words HDL interstitil lipoproteins * reverse cholesterol trnsport therogenic diet reverse cholesterol trnsport hs been obtined from in vitro studies in tissue culture (3-6). Additionlly, vrious models of interstitil fluid hve been used to provide in vivo informtion on the initil events ssocited with reverse cholesterol trnsport (reviewed in reference 7). Previous studies in this lbortory hve clerly shown tht HDL obtined from dog prenodl peripherl lymph, n ccepted model for interstitil fluid, differ in lipid nd polipoprotein composition nd in physicl chrcteristics from HDL obtined from plsm (8-1). In this report, we extend our studies of dog interstitil fluid HDL to include description of the differences between peripherl lymph nd plsm poa-i distribution s determined by nondenturing grdient gel electrophoresis-immunoblot nlysis (11). A mjor finding of this study is the identifiction of lipoprotein-unssocited poa-i with slow pre-bet electrophoretic mobility in control dog peripherl lymph despite its bsence in control dog plsm. METHODS Animls, diet nd peripherl lymph collection Mongrel dogs, free of hert worms, were fed either norml dog chow (five dogs) or dog chow supplemented with 1% sucrose,.3% cholic cid, 3% cholesterol, 2% lrd, nd.5% propylthiourcil (four dogs) for 3-4 dys (8). Low levels of plsm HDL re ssocited with incresed incidence of crdiovsculr disese (1). HDL re thought to exert their protective effects by fcilitting reverse cholesterol trnsport (2), the delivery of cholesterol from peripherl tissue to the liver for excretion. Much of the informtion concerning the role of HDL in Abbrevitions: HDL, high density lipoproteins; EDTA, ethylenedimine tetrcetic cid; DTNB, 5,5'-dithiobis (2-nitrobenzoic cid); CS, clf serum; BSA, b ine serum lbumin; po, polipoprotein; PBS, phosphte-buffered sline; LCAT, 1ecithin:cholesterol cyltrnsferse; LPL, lipoprotein lipse; PBST, phosphte-buffered sline contining.5% Tween 2. 'To whom correspondence nd reprint requests should be ddressed. Journl of Lipid Reserch Volume 29,

2 Both control nd cholesterol-fed dogs were fsted overnight prior to the surgery for lymph collection. The surgicl procedure for cute peripherl lymph collection ws crried out s previously described (8). Lymph smples were collected into chilled tubes contining EDTA, sodium zide, nd DTNB to provide finl concentrtions of O.l%, O.l%, nd 1.4 mm, respectively. Lymph ws collected over n 8-hr period t n verge lymph flow of 2 ml/hr per leg. On occsion, peripherl lymph ws collected from unnesthetized chroniclly cnulted dogs (Sloop, C. H., mnuscript in preprtion). In these instnces, the first hour's lymph collection ws discrded nd the second hour's collection ws used for the studies. Blood smples were drwn t the end of the experiment (or t the end of the second hour in chroniclly cnulted dogs) into tubes contining EDTA, sodium zide nd DTNB. Plsm ws obtined by centrifugtion t 15 g for 2 min t 4OC. Grdient gel electrophoresis-immunoblot nlysis The distribution of poa-i s function of lipoprotein size in both plsm nd peripherl lymph ws determined by grdient gel electrophoresis-immunoblot nlysis (described in detil in reference 11). Briefly, 16 fi1 of plsm or 8 pl of peripherl lymph were mixed with.25 vol of smple buffer (9 mm Tris, 8 mm boric cid, 4% sucrose,.1% bromphenol blue; ph 8.35) nd pplied cross the top of polycrylmide grdient gel (concve grdient) prepred in our lbortory. The top of ech gel ws verticlly prtitioned by plcing single 25-pl plstic well-former (obtined by cutting up 12-plce Phrmci plstic well-former) in the center of ech gel. This llowed us to electrophorese the plsm nd peripherl lymph smples from ech dog on the sme gel. Phrmci high moleculr weight stndrds were pplied in the 25-pl well. The smples were electrophoresed t 22 V for 24 hr t 1 C. After electrophoresis, strip of gel contining the moleculr weight stndrds ws excised from the center of the gel nd stined for protein. The remining two sections of the gel were incubted for 3 x 2 min in trnsfer buffer (2 mm Tris, 15 mm glycine; ph 8.4). The proteins in the gels were electrophoreticlly trnsferred onto chrge-modified nylon membrnes (Bio- Rd) t field strength of 3 V/cm for 24 hr t 1OC. Following 1-hr fixtion with.3% glutrldehyde, the trnsfers were blocked overnight t 5OoC with 1% clf serum (CS), 3% bovine serum lbumin (BSA),.1% sodium zide diluted in 5 mm sodium phosphte,.15 M NCl,.5% Tween 2; ph 7.4 (PBST). ApoA-1 ws loclized by 1-hr incubtion with got nti-dog poa-i ntiser diluted ppropritely in PBST with 1% CS. (The ntiser were monospecific s judged by immunoblots ginst both totl lipoproteins nd plsm subjected to SDS- PAGE.) This ws followed by five 1-min wshes in PBST, 1-hr incubtion with '251-lbeled ffinity-purified rbbit nti-got IgG (lo6 cpmitube diluted in PBST with 1% CS), nd finl wsh sequence. The dried trnsfers were utordiogrphed overnight t - 7OoC (Kodk XRP-5) nd subsequently scnned with Bio-Rd Model 62 video densitometer interfced to microcomputer. The utordiogrphic intensity ws converted to reltive poa-i mss through comprisons with stndrd contining known reltive concentrtions of trnsferred poa-i. These dt were then reduced to provide plot of percent poa-i distribution s function of RJ nd/or prticle size. Tretment of the glutrldehyde-fixed trnsfers with 1% NP-4 for 1 hr prior to immunolocliztion hd no significnt effect on the subsequent clcu- Ited distribution of poa-i, indicting tht ll epitopes were expressed eqully within the different lipoprotein popultions. Gel filtrtion chromtogrphy Plsm nd peripherl lymph were frctionted on 1.5 x 1 cm columns of Bio-Gel A-.5m. Prior to ppliction, smples of peripherl lymph were concentrted pproximtely tenfold under vcuum in Micro-Confilt (Biomoleculr Dynmics) pprtus. Two-ml smples were pplied to the column nd eluted with 5 mm sodium phosphte,.15 M NCl; ph 7.4 (PBS) with.1% EDTA nd.1% sodium zide t flow rte of pproximtely 12 ml/hr. Agrose immunoblots The electrophoretic mobility of the poa-i-contining prticles ws determined by grose electrophoresis (12) followed by immunoblotting. Smples of plsm, peripherl lymph, column frctions, or purified poa-i were electrophoresed in.5% grose contining.35% BSA. Electrophoresis ws crried out in buffer of 24 mm Tricine, 8 mm Tris,.1% clcium lctte; ph 8.6. Following electrophoresis, the grose strips were press-blotted onto nitrocellulose. The trnsfers were blocked with 3 % BSA in PBS for 1 hr t 37' C nd then incubted for 1 hr with monospecific got nti-dog poa-i ntiser diluted in PBS contining 1% BSA (PBS-BSA). The trnsfers were wshed three times with PBST nd then incubted for 1 hr with horserdish peroxidse-coupled rbbit ntigot IgG diluted in PBS-BSA. After three dditionl wshes, the trnsfers were developed with 4-chloro-lnpthol. Anlyticl methods Totl cholesterol ws determined enzymticlly (13) employing commercilly vilble kit (Behring Dignostics). ApoA-I concentrtions were determined by electroimmunossy (14) s previously described (15). Dt re presented s men f SEM. Sttisticlly significnt differences were evluted by Student's t-test or pired t-test where pproprite. 114 Journl of Lipid Reserch Volume 29, 1988

3 RESULTS Grdient gel electrophoresis-immunoblot nlysis ws employed to identify differences in poa-i distribution between plsm nd peripherl lymph of control, chow-fed dogs. Exmples of utordiogrms produced by this method re shown in Fig. 1 nd the normlized scns p- per in Fig. 2. Plsm poa-i could be loclized to t lest two distinct popultions of prticles within the HDL size rnge with modl dimeters of 1.4 *.9 nm nd 8.4 *.4 nm (n = 5). Hlf of the plsm poa-i (51.6 * 3.6%) could be loclized to prticles between 8. nd 9. nm (Fig. 3). Peripherl lymph poa-i distribution ws significntly different from tht of plsm. The mjority of HDLssocited poa-i could still be loclized to single pek between 8. nd 9. nm. However, in peripherl lymph, this mjor poa-i pek ws shifted to significntly smller modl dimeter (8.3 *.4 nm; P<.5 by pired t-test). When compred to plsm, reltively less peripherl lymph poa-i ws ssocited with lrger HDL prticles between 1. nd 12. nm (Fig. 3). Dimeter (nm) DOG 11 DOG 111 PP L L Std Fig. 1. Autordiogrms showing poa-i distribution in plsm (P) nd peripherl lymph (L) of two control dogs. Plsm cholesterol levels were 15 mg/dl nd 18 mg/dl for dogs 11 nd 111, respectively. (-) Indict- the reltive migrtions of protein size stndrds for ech set of gels. Numbers to the left re the Stokes dimeters for the protein size stndrds. The stndrd lne (Std) on the right ws used to convert utordiogrphic opticl density to reltive poa-i concentrtion. u) I- H Z 3 > [I I- n rn (I 4 1 DOG 11 e] DOG nm e 1. Fig. 2. Normlized poa-i distribution in plsm (open re) nd peripherl lymph (shded re) of the two control dogs depicted in Fig. 1. Autordiogrms were scnned nd the bsorbnces were converted to reltive mss of poa-i by procedures described in detil in reference 11. The distribution of poa-i in both plsm nd peripherl lymph for ech dog ws corrected to provide identicl res under ech curve. Dt re plotted s function of reltive migrtion of the gel (RJ) with the smllest prticles exhibiting the lrgest RJ. The clculted modl dimeters for the mjor peks re displyed with vlues for peripherl lymph ppering in itlics. The reltive migrtions of the protein size stndrds were: thyroglobulin (17. nm),.32; ferritin (12.2 nm),.43; lctte dehydrogense (8.16 nm),.65; nd humn serum lbumin (7.1 nm),.84. Popultions of poa-i prticles not evident in plsm were lso identified in peripherl lymph. In the HDL region of the gel, n dditionl minor poa-i-contining pek with modl dimeter of nm could be found (Fig. 2). Most striking, however,ws the ppernce of poa-i prticles in the smll pore region of the gel in peripherl lymph smples. These prticles, resolvble s one or sometimes two bnds (one shrp nd one brod), hd pprent dimeters smller thn both lbumin nd lipoprotein-unssocited poa-iv. Collectively, these lipoprotein-unssocited (i.e., not ssocited with clssicl lipoproteins) poa-i prticles comprised n verge of 14.7 * 3.8% (rnge; 6.5%-26.5%) of peripherl lymph poa-i. The presence of lipoprotein-unssocited poa-i ws confirmed by gel liltrtion column chromtogrphy (Fig. 4). Smples of peripherl lymph (Fig. 4, bottom), but not Rf Lcfcun, Sloop, nd Rohcim Chrcteriztion of dog prenodl peripherlymph lipoproteins 1141

4 1 :I * DIAMETER (nm) m Fig. 3. Distribution of poa-i s function of pprent prticle dimeter in control dog plsm (dshed line) nd peripherl lymph (solid line). Asterisks indicte significnt differences (P<.5 by pired t-test, n= 5) between plsm nd peripherl lymph percent poa-i distribution for given size intervl. plsm (Fig. 4, top), contined poa-i prticles eluting t volumes greter thn those for HDL. Grdient gel electrophoresis-immunoblot nlysis of these frctions confirmed tht this popultion of prticles ws composed of lipoprotein-unssocited poa-i (dt not shown). Mesurble mounts of cholesterol were not detected in the lipoprotein-unssocited poa-i column frctions. Smples of column frctions contining HDL nd lipoprotein-unssocited poa-i from plsm nd lymph were subjected to grose electrophoresis nd then immunoblotted for poa-i. ApoA-I from both plsm nd lymph HDL column frctions migrted with lph mobility (Fig. 5). In contrst, lipoprotein-unssocited poa-i from peripherl lymph migrted between LDL nd VLDL, i.e., with slow pre-bet mobility. The presence of slow pre-bet migrting poa-i ws lso demonstrted in grose immunoblots of unfrctionted peripherl lymph, but not plsm. ApoA-I immunoblots of plsm, lymph, nd column frctions subjected to isoelectric focusing did not revel ny significnt differences in poa-i isoform pttern (dt not shown). We next exmined the effect of n therogenic diet on the distribution of HDL-ssocited nd lipoproteinunssocited poa-i (Fig. 6 nd Fig. 7). A substntil portion of plsm poa-i ws still loclized to n HDL popultion between 8. nd 9. nm in size. However, the modl dimeter of this popultion ws significntly lrger (8.6.3 nm; P<.5) in dogs fed the therogenic diet when compred to controls (8.4 -fr.4 nm). As in control dogs, the corresponding poa-i HDL popultion in peripherl lymph ws significntly smller (8.3 -fr.3 - ln JJ.rl C 3 z L m L JJ.-I n L - H I I "1 PLASMA PERIPHERAL LYMPH ELUTION VOLUME (ml) t 2oo Fig. 4. Distribution of pd-i (A-A) nd cholesterol (m-m) in plsm nd peripherl lymph of control dog 11 following gel filtrtion chromtogrphy on Bio-Gel A-.5m. HDL pper s single pek eluting t volumes between 8 nd 1 ml. Lipoprotein-unssocited poa-i ppers s pek in peripherl lymph eluting t volumes between 1 nd 12 ml. - J W + v) too y 8 6 I 4 2 L 1142 Journl of Lipid Reserch Volume 29, 1988

5 HDL VLDL LDL 8 Origin I A B C D E F Fig. 5. Electrophoretic mobility of poa-i prticles in column frctions nd unfrctionted plsm nd peripherlymph of control dogs. A, Peripherlymph HDL; B, plsm HDL; C, peripherlymph lipoprotein-unssocited poa-i; D, plsm lipoprotein-unssocited poa-i; E, unfrctionted peripherl lymph; F, unfrctionted plsm. For HDL, column frctions eluting t volume of 9 ml were nlyzed. For lipoprotein-unssocited poa-i, column frctions eluting t volume of 11 ml were nlyzed. Unfrctionted plsm nd peripherl lymph smples were overloded nd thus do not disply clerly resolvble bnds. Nonetheless, the presence ofslow pre-bet migrting poa-i is clerly present in peripherl lymph but ppers to be bsent or substntilly reduced in plsm. The migrtion of humn lipoproteins is indicted. nm; P <.5 by pired t-test) thn the corresponding plsm smple. Additionlly, the difference in size of this popultion of poa-i prticles between plsm nd peripherl lymph in dogs fed the therogenic diet (.3 *.4 nm) ws significntly greter (P <.5) thn the difference found in dogs fed the control diet (.1 *.2 nm). In plsm, but not peripherl lymph, poa-i prticles substntilly smller (RJ.68-.8; nm) thn the min plsm HDL pek could lso be identified. In contrst to dogs fed the control diets, lipoproteinunssocited poa-i ws detected in both the plsm nd peripherl lymph of ll dogs fed the therogenic diet. The ppernce of lipoprotein-unssocited poa-i in both plsm nd peripherl lymph ws confirmed by gel filtrtion chromtogrphy (Fig. 8). Mesurble mounts of cholesterol were not ssocited with lipoprotein-unssocited poa-i in either plsm or peripherl lymph. Agrose immunoblots of the lipoprotein-unssocited frctions obtined from gel filtrtion chromtogrphy gin demonstrted tht both the plsm nd peripherl lymph poa-i frctions migrted with slow pre-bet mobility (dt not shown). Unfrctionted plsm nd peripherl lymph from cholesterol-fed dogs clerly contined poa-i prticles with slow pre-bet mobility (Fig. 9). These slow prebet poa-i prticles comigrted with the mjority of purified dog poa-i but hd slightly slower electrophoretic mobility thn both humn "pre-bet poa-i" nd purified humn poa-i. The degree of hypercholesterolemi ppered to substntilly influence the poa-i profile in both plsm nd peripherl lymph. In hyporesponding dogs (Fig. 7, dog 112), the popultion of lrger poa-i HDL prticles (modl dimeter, 1.9 nm) becme quite prominent in plsm, while in hyperresponding dogs (Fig. 7, dog 16), this popultion ws substntilly reduced. Furthermore, the reltive mount of plsm lipoprotein-unssocited poa-i in hyperresponding dogs ws elevted over tht found in hyporesponding dogs. This, however, ws not the cse in peripherl lymph where the reltive mount of lipoprotein-unssocited poa-i tended to decrese in hyperresponding dogs. Insted, with incresing degree of hypercholesterolemi, popultion of lrge prticles, (RJ.2-.5; nm) ccounted for n incresing percentge of the peripherl lymph poa-i. DISCUSSION Prenodl peripherl lymph is well-ccepted model for the study of interstitil fluid nd hs been used in investi- Dimeter (nm) DOG P m I12 L 3 DOG 16 P L 8 1 : Fig. 6. Autordiogrms showing poa-i distribution in plsm (P) nd peripherl lymph (L) of two hypercholesterolemic dogs. Dog 112 ws hyporesponder with plsm cholesterol vlue of 41 mg/dl. Dog 16 ws hyperresponder with plsm cholesterol vlue of 2435 mg/dl. (-) indictes the reltive migrtions of protein size stndrds for ech set of gels. Numbers to the left re the Stokes' dimeters for the protein size stndrds. Lcfcvre, Sloop, nd Roheim Chrcteriztion of dog prcnodl peripherl lymph lipoproteins 1143

6 7 I- H z 3 > U U I- H m U 4 DOG 112 DOG 16 Fig. 7. Normlized poa-i distribution in plsm (open re) nd peripherl lymph (shded re) of the two hypercholesterolemic dogs depicted in Fig. 6. Profiles were obtined s described in Fig. 2. The reltive migrtions of the protein size stndrds were: thyroglobulin (17. nm),.34; ferritin (12.2 nm),.44; lctte dehydrogense (8.16 nm),.67; nd humn serum lbumin (7.1 nm),.85. gtions concerning extrvsculr events ssocited with reverse cholesterol trnsport. Previously, we hve shown tht ll lipoprotein clsses nd polipoproteins present in dog plsm re lso present in dog peripherl lymph, lbeit t lower concentrtions (8, 9). It ws shown tht significnt differences exist between plsm nd peripherl lymph with respect to lipoprotein composition nd morphology which could not be explined by differentil trnsport of plsm lipoproteins cross the cpillry endothelium (8, 9). Of prticulr note ws the finding of reltively elevted HDL poe nd poa-iv content in peripherl lymph. The elevted HDL poe ws lter shown to be due to peripherl poe synthesis nd ssembly into discoidl HDL (16). Additionlly, evidence hd been previously presented suggesting tht plsm poa-icontining HDL undergo extrvsculr modifiction through enrichment with unesterified cholesterol (IO). In this report, we hve exmined more closely the extrvsculr modifictions of pd-i-contining HDL employing nondenturing grdient gel electrophoresis-immunoblotting methodology. Rf Dog plsm poa-i could be loclized to two mjor popultions of prticles with modl dimeters of 8.4 nm nd 1.4 nm. The smller popultion of prticles ccounted for the mjority of HDL poa-i nd is comprble in size to humn (HDL3)ggge while the lrger popultion is comprble in size to humn (HDL2b)gge(17). It is interesting to note tht dog HDL, which does not contin significnt mounts of poa-i1(18), exhibits size distribution similr to tht found for humn HDL Lp (A-I without A-11) which disply pprent dimeters of 8.4 nd 1.8 nm (19). The poa-i lipoprotein size distribution in peripherl lymph ws considerbly different when compred to tht of plsm. Most interesting ws the detection of poa-i in the smll pore region of the gel in smples of control dog peripherl lymph, but not in control dog plsm. Additionlly, in peripherl lymph, the reltive mount of poa-i in the 1.4-nm popultion ws decresed,.1-nm decrese in the modl dimeter of the smller 8.4-nm (plsm) HDL popultion occurred, nd n dditionl HDL pek ppered with modl dimeter of 9.3 nm. Severl fctors could ccount for these differences including: I) size-dependent differentil trnsport cross the - n L 4 n I d PLASMA PERIPHERAL LYMPH ELUTION VOLUME (ml) t-l t \ ol - 3 i -1 p u -Boo I J U W I- 1 2 Fig. 8. Distribution of poa-i (A-A) nd cholesterol (m-.) in plsm nd peripherl lymph of hypercholesterolemic dog 112 following gel filtrtion chromtogrphy on Bio-Gel A-.5m. HDL pper s single pek eluting t volumes between 8 nd 1 ml. Lipoproteinunssocited poa-i ppers s pek in plsm nd peripherl lymph eluting t volumes between 1 nd 125 ml Journl of Lipid Reserch Volume 29, 1988

7 A B C D E F G Fig. 9. Electrophoretic mobility of poa-i prticles in plsm nd peripherl lymph of hypercholesterolemic dog. Lnes A-E were subjected to immunoblot nlysis to loclize poa-i. Smples re s follows: A, hypercholesterolemic dog peripherl lymph; B, purified dog poa-i; C, hyperchlesterolemic dog plsm; D, purified humn poa-i; E, humn plsm with demonstrble 'pre-bet poa-i." Lnes F nd G were stined with oil red. Smples re: F, norml humn plsm; G, hypercholesterolemic dog plsm. cpillry endothelium; 2) peripherl polipoprotein synthesis; nd 3) extrvsculr modifiction of filtered plsm HDL vi uptke of desorbed cellulr lipids. The reltive decrese of the 1.4-nm prticle in peripherl lymph cn be prtilly explined by decrese in filtrtion cross the cpillry endothelium due to its lrger size. Crter, Joyner, nd Renkin (2) demonstrted tht n increse in effective protein rdius from 8.5 nm to 1.5 nm cn result in n pproximte 25% reduction in protein's lymph/plsm rtio. The reltive decrese of the 1.4-nm popultion (tken s the intervl between 1. nd 12. nm) is on the order of 4%. Thus, while n increse in size cn ccount for mjority of this decrese, we cnnot t present exclude the possibility tht other mechnisms, such s extrvsculr remodeling, re lso operting to specificlly reduce this HDL popultion in the peripherl lymph. The decrese in prticle size of the plsm 8.4-nm popultion to 8.3 nm in the peripherl lymph is not s esily explined. This smll difference in prticle dimeter ws extremely reproducible nd did not pper to be due to methodologicl problems. Smples of plsm nd lymph were electrophoresed side by side on the sme gel, thus eliminting potentil gel to gel vribility. The difference in size ws lso evident on precst Phrmci PAA 4/3 gels stined with oil red (dt not shown). This decrese in prticle size ppers to be t vrince with our own previously published dt (1) nd tht of others (21) which hve shown tht peripherl lymph lipoproteins in this size rnge (frction "L-11" in reference 1; HDLs in reference 21) tend to be lrger thn their plsm counterprts. Differences in methodologicl pproches my prtilly ccount for this pprent discrepncy. Assuming tht the peripherl lymph 8.3-nm prticle origintes from the filtered plsm 8.4-nm prticle, then the difference in size roughly trnsltes to moleculr mss difference of 8 Dltons. This difference is fr too smll to be cccounted for by the loss of n poa-i molecule nd is therefore most likely due to loss of lipid moieties. Since the dog hs no detectble cholesteryl ester trnsfer ctivity (22), loss of surfce lipids, especilly phospholipids (23) is likely explntion for the reduction in size. Within the HDL region of the gel, n dditionl popultion of poa-i-contining prticles with modl dimeter of 9.3 nm could be identified in peripherl lymph. Preliminry results (Lefevre, M., C. H. Sloop, nd P. S. Roheim, unpublished observtions) hve shown tht poe cn lso be loclized to discrete popultion of prticles which migrtes to the sme position on the gel s the 9.3-nm poa-i prticle. Whether in fct both poe nd po A-I reside on the sme 9.3-nm prticles remins to be estblished. As previously stted, of prticulr interest ws the observtion tht significnt portion of peripherl lymph poa-i ws invribly present in the smll pore region of the gel (lipoprotein-unssocited poa-i). Gel filtrtion chromtogrphy confirmed the presence of the lipoprotein-unssocited poa-i prticles in peripherl lymph nd their bsence in control dog plsm. The presence of lipoprotein-unssocited poa-i in peripherl lymph is not unique to the dog model. In preliminry studies we hve been ble to demonstrte the presence of lipoprotein-unssocited poa-i in rbbit prenodl peripherl lymph (Lefevre, M., C. H. Sloop, nd P. S. Roheim, unpublished observtion). Additionlly, in humn lymphedem fluid, Reichl nd ssocites (21) demonstrted by gel filtrtion chromtogrphy, the presence of smllsized (smller thn lbumin) poa-i prticles. On grose electrophoresis, the lipoprotein-unssocited poa-i frction migrted with slow pre-bet mobility. Low moleculr weight, pre-bet-migrting poa-i-contining prticles hve lso been identified in normolipidemic nd hyperlipidemic humn plsm (24-26). Similr to wht ws observed in dog peripherl lymph, humn plsm pre-bet migrting poa-i exhibited size heterogeneity on nondenturing grdient gels (26). Dog slow pre-bet poa-i ws found to comigrte on grose electrophoresis with the mjority of purified dog poa-i. Similrly, in our hnds, humn prebet poa-i ws lso found to comigrte on grose electrophoresis with purified humn poa-i. Tken together, the bove observtions suggest tht dog slow pre-bet poa-i nd humn pre-bet poa-i re physiologiclly homologous. In humn plsm, pre-bet migrting pd-i ws found to contin pproximtely 1% lipid, including free cholesterol, esterified cholesterol, nd phospholipids (24). We could not detect the presence of cholesterol (free or es- Lcfcun, Sloop, nd Roheim Chrcteriztion of dog prenodlperipherl lymph lipoproteins 1145

8 terified) in ssocition with the lipoprotein-unssocited poa-i. However, this my be due to lck of sufficient mteril for dequte nlysis. Attempts t further lipid chrcteriztion following specific immunoprecipittion of the lipoprotein-unssocited poa-i column frction were not successful. We could not unequivoclly demonstrte the presence of specific phospholipids or other lipids in ssocition with the lipoprotein-unssocited poa-i due to the presence of contminting lipids in the IgG preprtion. However, given the heterogeneous migrtion of lipoprotein-unssocited poa-i on nondenturing grdient gels, it is likely tht this poa-i frction is complexed with minor mounts of lipids or possibly low moleculr weight polipoproteins. Three mechnisms cn be envisioned for the production of lipoprotein-unssocited poa-i in the peripherl lymph: I) peripherl synthesis; 2) fusion of poa-1- contining HDL; nd 3) displcement of HDL poa-i by phospholipids, free cholesterol, or other polipoproteins derived from either peripherl cells or from the lipolysis of triglyceride-rich lipoproteins. Newly synthesized poa-i hs been found in the lipoprotein-unssocited frction in cultures of Hep G2 cells (27, 28) nd following trnsfection of humn poa-i gene into 3T3 cells (29). While poa-i synthesis hs been demonstrted in the peripherl tissue of vin species (3), there hs been no evidence for peripherl poa-i synthesis in mmmlin species despite the presence of peripherl poa-i mrna (31). Severl studies hve shown tht lipoprotein-unssocited poa-i cn be produced following the fusion of model HDL prticles contining two poa-i molecules per prticle to form product HDL contining three poa-i molecules per prticle (23, 32). Fusion ppers to tke plce following reduction in the reltive mount of surfce components on n HDL prticle s would occur following the LCAT-ctlyzed production of core cholesteryl esters from the surfce lipids, phosphtidylcholine nd free cholesterol. However, in peripherl lymph, LCAT ctivity is quite low (33) nd the lipoproteins re enriched rther thn deficient in surfce lipids (9). The enrichment of HDL surfce lipids rgues for displcement type of mechnism for the formtion of lipoprotein-unssocited poa-i in the peripherl lymph. Tll et l. (34), Nichols et l. (35), nd Forte et l. (36) hve demonstrted tht phospholipids cn trnsfer to HDLZ, resulting in the displcement of poa-i from the surfce. Additionlly, it hs been estblished tht certin polipoproteins, most notbly poa-11, cn lso displce poa-i from the HDL surfce (18). On the other hnd, HDL pper to hve substntil cpcity to incorporte free cholesterol without pprent disruption of prticle integrity (37). These surfce components hve two possible origins. Following lipolysis of VLDL nd chylomicrons, surfce remnnts re produced which re subsequently incorported into HDL (38-4). Given the physicl prox- imity between the sites of lipolysis nd the interstitil fluid nd the reltively low concentrtion of HDL in the interstitil fluid (leding to high rtio of surfce remnnts to HDL), lipolysis of triglyceride-rich lipoproteins my provide sufficient surfce components to led to the displcement of peripherl lymph HDL poa-i. Indeed, one report hs demonstrted the production of plsm lipoprotein-unssocited poa-i following heprin-enhnced lipolysis of plsm triglyceride-rich lipoproteins (25). HDL cn lso become enriched in surfce lipids fter interction with lipids desorbed or secreted from peripherl cells. This process, which is regrded s the initil step in reverse cholesterol trnsport, hs been extensively studied in cell culture systems. It is well documented tht HDL cn tke up cellulr free cholesterol, leding to net cholesterol efflux (3, 4). There re less extensive experimentl dt showing tht HDL polipoproteins cn promote the net efflux of cellulr phospholipids (3). Perhps more importnt, peripherl cells lso hve the bility to secrete poe which, when isolted, ppers in the form of phospholipid-contining discoidl structures (41). These poe-contining discoidl structures would be free to combine with peripherl lymph poa-i-contining HDL (42) nd possibly led to poa-i displcement. Conclusions similr to these hve been mde by Riechl et l. (21) with respect to the production of lipoprotein-unssocited poa-i in humn lymphedem fluid. A previous study demonstrted tht humn plsm lipoprotein-unssocited poa-i levels re elevted in hyperlipidemi (25). A more recent study showed similr results for humn plsm pre-bet migrting poa-i (26). We employed high cholesterol, thyroid-suppressing diet to induce hypercholesterolemi in dogs. Consistent with the observtions mde in hyperlipidemic humns, hypercholesterolemic dog plsm contined lipoprotein-unssocited poa-i with slow pre-bet mobility. Furthermore, the mount of plsm lipoprotein-unssocited poa-i ws elevted in hyperresponding dogs over tht found in hyporesponding dogs. However, contrry to wht ws expected, peripherl lymph lipoprotein-unssocited poa-i did not increse with incresing degree of hypercholesterolemi. Insted, the proportion of lipoproteinunssocited poa-i in peripherl lymph ws lower in hyperresponding dogs thn in hyporesponding dogs. The lower level of lipoprotein-unssocited poa-i in the hyperresponding dogs ppered to coincide with the ppernce of new popultion of lrge-sized poa-i prticles. These observtions mde in peripherl lymph of cholesterol-fed dogs cn be prtilly explined by the findings of Nichols et l. (35) who found tht the type of products formed following incubtion of HDL with phospholipid vesicles is dependent on the rtio of the two components. At low phospholipid to HDL rtios, lipoprotein-unssocited poa-i is produced; t high rtios, lrge discoidl 1146 Journl of Lipid Reserch Volume 29, 1988

9 poa-i complexes re formed t the expense of the lipoprotein-unssocited poa-i product. In the hypercholesterolemic dog, plsm nd peripherl lymph HDL poa-i levels re less thn 25% of control vlues (8). In the fce of constnt or elevted cellulr phospholipid efflux (possibly in the form of poe-contining discoidl prticles), this reduction in peripherl lymph HDL levels would increse the phospholipid to HDL rtio nd possibly result in the formtion of lrge poa-i discoidl structures. This would be consistent with the observtion of discoidl lipoproteins contining poa-i both with nd without poe in the peripherl lymph of hypercholesterolemic dogs (1). If the lrge poa-i prticles in the peripherl lymph re discoidl in nture, then their suitbility s substrtes for LCAT (contining both free cholesterol nd poa-i) would most likely result in their trnsformtion into sphericl prticles with smller pprent dimeters. This would ccount for their bsence in the plsm comprtment. It is cler from these studies tht the reltionship between plsm lipoproteins nd their counterprts in the extrvsculr spce is complex. Extrvsculr HDL do not just simply gin cholesterol, but undergo number of trnsformtions tht my include lipid cquisition prticle fusion, nd loss of poa-i. Becuse these prticles re bsent in the plsm, these peripherlly modified HDL re either rpidly removed from the circultion or quickly modified by LCAT, LPL, lipid trnsfer proteins, ndlor heptic lipse. Given tht the mgnitude of cholesterol trnsport from the periphery to the liver hs been estimted to be between.45 nd.9 gldy in n dult mle (43), the extrvsculr remodeling of HDL ssocited with reverse cholesterol trnsport is likely to hve significnt impct on plsm HDL specition nd subclss distribution. I The uthors re grteful to Ms. J. Goudey-Lefevre, Ms. C. Tte, nd Ms. C. Tierney for their technicl ssistnce, nd to Ms. L. Lehmnn nd Ms. M. A. Benoit for their editoril ssistnce. 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10 plsm high density lipoprotein subpopultions contining polipoprotein A-I without polipoprotein A-11. J Lipid Res. 28: Kunitke, S. T., K. J. L Sl, nd J. P. Kne Apolipoprotein A-I-contining lipoproteins with pre-bet electrophoretic mobility..i. Lipid Res. 26: Derr, W. H., U. Minzlff, nd H. Gretn Quntittive determintion of polipoprotein A-I in high-density lipoproteins nd free polipoprotein A-I by two-dimensionl grose gel lipoprotein- rocket immunoelectrophoresis of humn serum. Biochim. Biophys. Act. 879: Ishid, B. Y., J. Frolich, nd C. Fielding Prebetmigrting high density lipoprotein: quntittion in norml nd hyperlipidemic plsm by solid phse rdioimmunossy following electrophoretic trnsfer. J. Lipid Res. 28: Thrift, R. N., T. M. Forte, B. E. Choon, ndv. G. Shore Chrcteriztion of lipoproteins produced by the humn liver cell line, Hep G2, under defined conditions. J. Lipid Res. 27: Forte, T. M., A. V. Nichols, J. Selmek-Hlsey, L. Cylor, nd V. G. Shore Lipid-poor polipoprotein A-I in Hep G2 cells: formtion of lipid-rich prticles by incubtion with dimyristoylphosphtidylcholine. Biochim. Biophys Act. 92: Lmon-Fv, S., J. M. Ordovs, G. Mndel, T. M. Forte, R. H. Goodmn, nd E. J. Schefer Secretion of polipoprotein A-I in lipoprotein prticles following trnsfection of the humn polipoprotein A-I gene in 3T3 cells. J. Bid. Chem. 262: Shckelford, J. E., nd H. G. Lebherz Synthesis nd secretion of polipoprotein A1 by chick brest muscle. J. Bid. Chem. 258: Sorci-Thoms, M., L. L. Rudel, nd D. L. Willims Peripherl polipoprotein A-I mrna bundnce in the non-humn primte. Arteriosclerosis. 6: Nichols, A. V., P. J. Blnche, E. L. Gong, V. G. Shore, nd T. M. Forte Moleculr pthwys in the trnsformtion of model discoidl lipoprotein complexes induced by 1ecithin:cholesterol cyltrnsferse. Biochim. Biophys. Act. 834: Dory, L., C. H. Sloop, L. M. Boquet, R. L. Hmilton, nd P. S. Roheim Lecithin:cholesterol cyltrnsfersemedited modifiction of discoidl peripherl lymph high density lipoproteins: possible mechnism of formtion of cholesterol-induced high density lipoproteins (HDL,) in cholesterol-fed dogs. Proc. Ntl. Acd. Sci. USA. 8: 348Y Tll, A. R., V. Hogn., L. Askinzi, nd D. M. Smll Interction of plsm high density lipoproteins with dimyristoyllecithin multilmellr liposomes. Biochemisty : Nichols, A. V., E. L. Gong, T. M. Forte, nd P. J. Blnche Interction of plsm high density lipoprotein HDL,b (d glml) with single-bilyer liposomes of dimyristoylphosphtidylcholine. Lip&. 13: Forte, T. M., C. L. Ren, R. W. Nordhusen, nd A. V. Nichols Formtion of phospholipid-rich HDL: model for squre-pcking lipoprotein prticles found in interstitil fluid nd in betlipoproteinemic plsm. Biochim. Biophys. Act. 834: Jons, A., L. K. Hesterberg, nd S. M. Drengler Incorportion of excess cholesterol by high density serum lipoproteins. Biochem. Biophys. Act. 528: Eisenberg, S., nd T. Olivecron Very low density lipoprotein. Fte of phospholipids, cholesterol, nd polipoprotein C during lipolysis in vitro. J. Lipid Res. 2: Tll, A. R., P. H. R. Green, R. M. Glickmn, nd J. W. Riley Metbolic fte of chylomicron phospholipids nd poproteins in the rt. J. Clin. Inuest. 64: Schefer, E. J., M. G. Wetzel, G. Bengtsson, R.. Scow, H. B. Brewer, Jr., nd T. Olivecron Trnsfer of humn lymph chylomicron constituents to other lipoprotein density frctions during in vitro lipolysis. J. Lipid Res. 23: Bsu, S. K., Y. K. Ho, M. Brown, D. W. Bilheimer, R. G. W. Anderson, nd J. L. Goldstein Biochemicl nd genetic studies of the poprotein E secreted by mouse mcrophges nd humn monocytes. J. Bid. Chem. 257: Gordon, V., T. L. Innerrity, nd R. W. Mhley Formtion of cholesterol- nd poprotein E-enriched high density lipoproteins in vitro. J Biol. Chem. 258: Stein, O., Y. Stein, M. Lefevre, nd P. S. Roheim The role of polipoprotein A-IV in reverse cholesterol trnsport studied with cultured cells nd liposomes derived from n ether nlog of phosphtidylcholine. Biochim. Bio- phys. Act. 878: Journl of Lipid Reserch Volume 29, 1988