In Vitro and In Vivo Interactions of Triton 1339 with Plasma Lipoproteins of Normolipidemic Rhesus Monkeys

Transcription

1 n Vitr and n Viv nteractins f 1339 with Plasma Lipprteins f Nrmlipidemic Rhesus Mnkeys Preferential Effects n High Density Lipprteins Kysuke Yamamt, Betty Shen, Christpher Zarins, and Angel M. Scanu WR-1339 was incubated in vitr in varius prprtins with plasma frm nrmlipidemic rhesus mnkeys r with ultracentrifugally purified lipprteins, and the prducts were examined by ispycnic density gradient ultracentrifugatin, agarse clumn chrmatgraphy, electrphretic and immunchemical techniques, and electrn micrscpy. Sme experiments used ap A-l, ap A-ll, r labeled with either 125 r 131. At cncentratins f less than 1 mg/ml plasma, interacted preferentially with HDL, changing lipprtein size and density; was prgressively incrprated int the HDL particles, displacing ap E, ap A-l, and ap A-ll. At cncentratins abve 1 mg/ml plasma, displaced all ap A-l frm the particle, and much lipid was disslved int the micelles. When -treated HDL particles were used as a substrate fr the enzyme LCAT, enzyme activity decreased in parallel t the displacement f ap A-l. There was n displacement f ap B frm LDL nr any lss f lipids; but the particles became defrmed and frmed ruleaux. A single intravenus dse f WR-1339 administered t a nrmlipidemic mnkey (N) and t a hyperchlesterlemic mnkey (H) resulted in cncentratindependent HDL changes similar t thse bserved in vitr. LDL was less affected by, with changes ccurring nly at high dses. After these structural changes, intravenusly injected 131 ap A-l disappeared rapidly frm the circulatin; 125 ap A- disappeared less rapidly. These increased clearances were accmpanied by a drp in ap A-l plasma levels and the disappearance f HDL particles frm plasma. The lipprtein and aplipprtein patterns returned t nrmal 14 days after. We cnclude that WR-1339, when expsed t rhesus plasma in vitr r in viv, interacts preferentially with HDL in a dse-dependent manner. At lw cncentratins, acts n surface cmpnents f the HDL particle; at higher cncentratins, penetrates the particle, causing structural disruptin. Because f its high affinity fr HDL, WR-1339 is a useful reagent fr study f HDL structure-functin relatinships. (Arterisclersis 4: , July/August 1984) S ince the first reprt n the prductin f hyperlipidemia by the intravenus administratin f WR-1339 in experimental animals, 1 this detergent has been used as a tl fr studying lipid Frm the Departments f Medicine, Bichemistry, Biphysics, Theretical Bilgy, and Surgery, University f Chicag, Chicag, llinis. A preliminary accunt f this wrk has appeared in abstract frm in Arterisclersis 1982;2:445a, as submitted t the 36th Annual Meeting f the Cuncil n Arterisclersis, Dallas, Texas, Nvember This research was supprted by U.S. Public Health Service Grants HL and HL Address fr reprints: Dr. Angel M. Scanu, Department f Medicine, Bx 231, University f Chicag, 5841 Suth Maryland Avenue, Chicag, llinis Received Nvember 7,1983; revisin accepted March 7, metablism. 2 " 15 Earlier studies in the dg led t the recgnitin f a direct effect f WR-1339 n the physicchemical prperties f the plasma lipprteins bth in vitr and in viv. 12 ' 13 n thse and in the later studies in the squirrel mnkey 11 and the rat, 15 was shwn t disrupt the high density lipprtein (HDL) particles and t cause a dissciatin between apprteins and lipid cmpnents. Because f the apparent high affinity f WR-1339 fr HDL and the recgnized rles that this lipprtein class plays in lipid metablism, 16 we elected t further elucidate the mechanism f the interactin f with HDL, with an emphasis n the mlecular basis f these interactins. The purpse f this manuscript is t reprt studies n the in vitr events attending the interactin f WR-1339 with rhe-

2 TRTON WR 1339 AND PLASMA LPOPROTENS Yamamt et al. 419 sus mnkey plasma and lipprteins using a cmbinatin f physical, chemical, and immunlgical methds, aided by the use f radiactive cmpunds. We als fllwed the changes in plasma lipprteins and aplipprteins in their prgressin and regressin phases fllwing a single intravenus administratin f WR-1339 int ne nrmchlesterlemic mnkey and ne animal made hyperchlesterlemic by the administratin f a diet cntaining 25% ccnut il and 2% chlesterl. The results btained are the subjects f this reprt. t will be shwn that there is a clse parallel between plasma lipprtein changes induced by in viv and in vitr. Methds Materials Fr the in vitr studies, bld was cllected by venus puncture int tubes cntaining.1 M EDTA as an anticagulant frm tw fasting, healthy male rhesus mnkeys weighing 13 t 15 kg, which were fed regular Purina Chw. Their plasma chlesterl levels were between 18 and 13 mg/dl and the triglycerides, between 18 and 42 mg/dl. Fr the in viv studies, tw male adult rhesus mnkeys (bdy weight 13 t 15 kg, 13 years ld) were used. One was fed a regular Purina Chw diet (Ralstn Purina Chw Cmpany, St. Luis, Missuri), the ther, a diet f 7.5% mdified, lw fat Purina Primate Chw supplemented with 25% ccnut il, 2% chlesterl, 1% vitamin mix, and 1.5% gelatin. These mnkeys were maintained n their respective diets fr a perid f 6 mnths. All f the experiments cnfrmed with institutinal guidelines n animal care. The mnkeys fasted at least 16 hurs befre the experiments began. They were sedated with 5 t 7 mg Ketaset i.m. (Ketamine HC, Bristl Labratries, Syracuse, New Yrk) per kg f bdy weight. The idinated hmlgus aplipprtein A-l and A-ll (each 2 /xci) were injected 3 minutes befre injectin with a 2% slutin f WR-1339 (Tylxapl, Sigma) in.5 M phsphate buffer, ph 7.2 (6 mg/kg/bdy weight). This dse f was purpsefully chsen because it was smaller than thse previusly used in experimental animals; 24 ' 13 in preliminary experiments, this dse was sufficient t induce physicchemical changes in plasma lipprteins withut causing hemlysis. We cnducted three independent experiments in each mnkey at apprximately 3-mnth intervals. n each case, we btained similar results n plasma lipprteins, aplipprteins, and lipids. Thus, nly representative experiments will be described. Bld samples (4 ml) were withdrawn frm the femral vein immediately befre and after intravenus injectin f at, 15, 6, 18, and 27 minutes and at 1,3, 7,1, and 14 days. The samples were cllected in.1 M EDTA and the plasma was rapidly separated by lw speed centrifugatin ( C). ncubatin Cnditins n Vitr WR-1339 was disslved in.5 M phsphate buffer (ph 7.2). Rhesus plasma was incubated with in 1:1 rati (vl/vl) s that the incubatin mixture cntained at cncentratins ranging frm t 1 mg/ml. n incubatins f with separated lipprteins, the prtein cncentratin was adjusted t be similar t the HDL cncentratin in whle plasma/ mixtures. The incubatin was cnducted at 37 C fr 2 hurs based n the results f preliminary bservatins. Ultracentrlfugal Fltatin Lw density lipprteins (LDL) f d = 1.19 t 1.63 g/ml and high density lipprteins (HDL) f d = 1.63 t 1.21 g/ml were separated by ultracentrifugal fltatin and washed free f ther plasma prteins accrding t prcedures established in this labratry. 17 ' 18 They were extensively dialyzed at 4 C against.15 M NaC, 1" 3 M EDTA (ph 7.2) befre use. Density Gradient Ultracentrlfugatln After incubatin, the reacted mixture was separated by a single-step density gradient ultlracentrifugatin using a technique previusly described. 19 The effluents were cntinuusly mnitred at 28 nm in an SCO UA-5 (nstrumentatin Specialty Cmpany, Omaha, Nebraska) and cllected as.4 ml fractins. Quantitative mmunassay f Apprtelns Purified rhesus ap A-l and A-ll were btained as previusly described. 2 Rhesus ap B was btained frm nrmal rhesus serum accrding t the methd f Karlin et al. 21 Rhesus ap E was purified frm hyperchlesterlemic rhesus ap very lw density lipprtein (VLDL) by high perfrmance liquid chrmatgraphy. 21 Antisera against either ap A-l, B, r E were raised in New Zealand rabbits by injecting 1 /ug f each antigen emulsified in Freund's adjuvant intramuscularly three times at intervals f 2 weeks. The first injectin cntained cmplete Freund's adjuvant; the ther tw, incmplete Freund's adjuvant. The immunassays fr rhesus ap A-l, B, and E were carried ut by the rcket immunelectrphretic prcedure described by Laurell. 23 Fr plasma samples and lipprteins cntaining, n specific treatment was necessary t insure maximal antigenicity. n the samples nt cntaining the detergent, ap A-l was determined in the presence f 7 M urea; ap B and E were determined in the presence f.1% X-1. Radlldlnatln f Ap A-l, Ap A-ll, and WR-1339 Ap A-l and A-ll were idinated with either 125 r using the lactperxidase methd. 24 The radilabeled prtein was separated frm unreacted 12S Nal

3 42 ARTEROSCLEROSS VOL 4, N 4, JULY/AUGUST 1984 r 131 Nal in a clumn packed with Sephadex G-75 superfine. WR-1339 in.5 M phsphate buffer (ph 7.2) was labeled with 125 by the methd f McFarlane. 25 Free idine was remved frm the radilabeled prtein by passage thrugh a Sepharse 4B clumn. Radilabellng f HDL n Viv Thirty minutes after the intravenus injectin f 3 /nci f bth 131 ap A-l and 125 ap A-ll fr a rhesus mnkey, bld was btained. HDL f d = g/ml cntaining the tw radiactive apprteins was separated by ultracentrifugal fltatin and dialyzed against.15 M NaC, 1-3 M EDTA (ph 7.2) befre use. Mlecular Sieve Chrmatgraphy Gel filtratin f the incubated mixtures f lipprteins and WR-1339 was cnducted in glass clumns (2.5 x 7 cm) packed with Sepharse 4B (Pharmacia Fine Chemicals, Upsala, Sweden). The clumns were eluted with.5 M phsphate (ph 7.2) at a flw rate f 2 ml/hr at 6 C. Eluates were mnitred at 28 nm. Electrn Micrscpy Fractins frm single spin ultracentrifugatin were negatively stained with 1% sdium phsphtungstate (ph 7.) after depsitin nt a carbn filmcated cpper grid. The specimens were examined in a Phillips EM 3 micrscpe with cndenser and bjective apertures f 1 ptm and 5 ^m, respectively. The acceleratin vltage was 8 kv, and all specimens were examined at 55, magnificatin. Average diameters in A were btained frm the sizing f 1 particles. Chemical Analysis Befre analysis, all samples were extensively dialyzed against.5 M NH 4 HCO 3 buffer (ph 8.2). The prtein cntent was determined by the Lwry prcedure, 26 mdified t include the additin f.5% sdium ddecyl sulfate t reagent A. Ttal and free chlesterl were determined enzymatically accrding t a slight mdificatin f the prcedure f Allain et al. 27 and Gall et al. 28 Lipid phsphrus was measured accrding t the methd f Bartlett. 29 Ttal triglycerides were determined in a Technicn Autanalyzer after isprpanl extractin f Zelite-treated samples. was measured after extractin with 1 vlumes f isprpanl at 278 nm. 6 Electrphretic separatin f apprteins was perfrmed in plyacrylamide gels cntaining 8 M urea 3 r.1 % SDS. 31 Agarse gel electrphresis was carried ut n Agarse Universal Electrphresis film (AC-Crning, Pal Alt, Califrnia). After electrphresis, lipprteins were fixed and stained with either Fat Red 7B r Amid Black 1B. Gradient Gel Electrphresis Gradient gel electrphresis was carried ut n a Pharmacia Electrphresis Apparatus GE-4 laded with gradient gels PAA 4/3 at 14 C at 125 V fr 2 hurs. Fllwing electrphresis, the gels were fixed and stained vernight in.4% Cmassie Brilliant Blue G-25 in 3.5% perchlric acid fllwed by destaining in 5% acetic acid. Mlecular weight standards were run in each gel slab. Determinatin f LCAT Activity n Vitr LCAT activity was determined by the mdified methd f Stkke and Nrum. 32 Reagents All chemicals were reagent grade. WR was purchased frm Sigma Chemical Cmpany, St. Luis, Missuri. Thyrglbulin, Catalase and Aldlase (Pharmacia Fine Chemicals, Piscataway, New Jersey) were used as the mlecular weight standards. Na 125 l and Na t31 l (carrier-free) were purchased frm Amersham Crpratin, Arlingtn Heights, llinis. Radiactivity measurements were carried ut in a Tracr Analytic Mdel 119 autmatic gamma-cunter (Tracr Analytic, Elk Grve Village, llinis). Results n Vitr Studies Effect f n Whle Plasma, HDL and LDL: Analyses by Agarse Gel Electrphresis The effect f was dse-dependent. n the case f whle plasma (Figure 1 A), as increased in cncentratin, the lipid-stained band in the a, psitin decreased in mbility; at a cncentratin f 5 mg/ml, the plasma remained clse t the rigin. The gels stained with Amid Black 1B (prtein stain) shwed that, the a, band decreased in mbility t the 6 regin (Figure 1 A, B), a regin where purified A-l was fund t mve in ther experiments (data nt shwn). When 12S was used as a tracer, the peak f radiactivity at 5 mg was in the same area as the lipid band (Figure 2) indicating that had caused a dissciatin f the lipids frm the serum carriers and particularly frm the a, lipprteins. This interpretatin was supprted by the studies f -HDL mixtures (Figure 1 B) shwing that the detergent caused the lipid-stained band t mve away frm the HDL prtein. At 1 mg f, this band was behind the rigin, whereas the prtein band was in the a, psitin. Cntrary t HDL, the LDL band shwed nly a decrease in electrphretic mbility even at high cncentratins f (Figure 1 C).

4 TRTON WR 1339 AND PLASMA LPOPROTENS Yamamt et al. 421 Lip Rrt spycnic Ultracentrifugal Prfiles f Rhesus Plasma fllwing ncubatin with ( mg/ml) As shwn in Figure 3, in the cncentratin f 2 t 1 mg/ml f plasma caused a marked change in the 28 nm single-spin prfile. The band crrespnding t HDL (Peak a) was prgressively shifted t lighter densities with an attending increase in absrbance readings at 28 nm due t bth (maximal absrbance f, 278 nm) and prtein. We have named these bands; b (2 mg ); c,d (5 mg ); and e,f,g (1 mg ) with buyant densities varying between 1.63 and g/ml (Table 1). (mg/fnl) ex 1 4 \r -2-4 B OJO 8 t t-i (-H (+) fi A CM c Figure 1. Agarse gel electrphretic patterns f whle plasma (A), HDL 2 (B), and LDL (C) after incubatin with varius cncentratins f WR-1339 fr 2 hurs at 37 C; HDL 2 (3 mg prtein/ml), and LDL (2 mg prtein/ml). After incubatin, 3 ^l f each sample was applied in duplicate t the agarse film. Fr details see text. After electrphretic separatin, ne sample was stained with Fat Red 7B and the ther with Amid Black 1B. Up = lipid staining; prt = prtein staining; = rigin. The arrws indicate the psitin f the new band behind the a, regin. ) ' ' ' ' '"(+) Distance (cm) Figure 2. Electrphretic distributin f 12S l-labeled after incubatin with whle plasma. Tracer amunts f 12S l-labeled were mixed with unlabeled in the stated cncentratins. After incubatin with whle plasma, 3 tx\ aliquts f each mixture were applied in duplicate t agarse films. One sample was stained with Fat Red 7B, the ther was cut int.5 cm segments and cunted.

5 422 ARTEROSCLEROSS VOL 4, N 4, JULY/AUGUST 1984 mg 2 mg 5 mg 1 mg Absrbanc* - 6 ' (28nm).3- c 9 n n n s i i» t a s 1 t, B 1 d ap E (pg/hil) ap B ( itetii,. il ) t ( cpm) ( ra 'l)apa- (cpm) (cpm) + y\ 5O5 2O253O Fra c t i n Nu mbtr. h r-r-, r h r-r, p b r i. 1 1 UB LO4 L6 UO J JO US U3 14 LOG UO US L3 1.4 L6 JO U5 Density (g/ml) Figure 3. Distributin f ap A-, ap E, ap B, 12s l-labeled, 131 l-labeledapa-l,and 12S l-labeled ap A-ll in ultracentrifugal fractins f plasma incubated with varius cncentratins f (2 hurs, 37 C). The labeling f plasma with 131 l-ap A- r 125 l-ap A-ll was carried ut in viv (see Methds). One mililiter f each incubated sample was separated by single-spin density ultracentrifugatin. The effluents were cntinuusly mnitred at 28 nm in an SCO UA-5 mnitr and.4 ml fractins were cllected fr a ttal f 32 fractins. The cncentratins f ap A-, ap E, and ap B were quantified by electrimmunassay. The amunt f radiactivity in each tube was determined. The pled HDL fractins were arbitrarily named frm a thrugh g (a = fractin number 18-26; b = 15-23;c = 11 18; d = 23-26;e = 11-18; f = 19-22; g = 23-26). Table 1. Physlcchemlcal Prperties f the Main Peaks (a-g) Shwn n Figure 3 Peak a b c d e f Diameter (A) Hydrated density (g/ml) Chemical cmpsitin (weight%) Prtein Ttal chlesterl Phsphlipid Triglyceride Diameter was btained by gradient gel electrphresis g

6 TRTON WR 1339 AND PLASMA LPOPROTENS Yamamt et al. 423 Frm the analysis f the mg clumn in Figure 3, it is evident that authentic HDL cntained essentially ap A-l and ap A-ll, little ap E, and n ap B, and that banded in the same density range as HDL. At 2 mg f, there was sme displacement f ap A-l frm HDL, whereas all f the ap E was in the bttm fractin. There were n detectable effects n ap B. At 5 mg f /ml plasma Peak C cntained neither ap A-l (which was all fund in Fractin d) nr ap E, and cnsisted f, ap B, and ap A-ll. At 1 mg /ml plasma, three fractins were generated: Fractin e cntained and ap B, while Fractins f and g cntained ap A-l, ap A-ll, and. n terms f apprteins, Fractin f cntained mainly ap A-ll and Fractin g cntained ap A-l. Thus, there was a marked difference in the effect f n ap A-l, ap A-ll, ap E, and ap B as a functin f the amunt f detergent added t plasma. Except fr ap B, this detergent displaced the aplipprteins fr the HDL surface, althugh t a different degree. The plypeptide distributin f Fractins a-g f the single spin prfiles f Figure 3 as assessed by SDS and 8 M urea-plyacrylamide gel electrphresis crrbrated the data btained by the immunchemical r radiactivity measurements (Figure 4). The general prperties f Fractins a-g are shwn in Table 1. Additin f up t 2 mg/ml caused an increase in the size f the HDL- cmplex which retained the same lipid cntent and cmpsitin as authentic HDL althugh underging a prgressive lss f apprteins (see data n Peaks a, b, and c). The ther peaks (d-g) cntained mainly and apprteins. Mre than 9% f the that was present in the ultracentrifuged plasma was recvered in the majr and minr peaks. By electrn micrscpy (Figure 5), the cntrl HDL had a diameter f 15 ± 1 A, and Peak b cntained spherical particles with a diameter f 125 ± 15 A. The particles crrespnding t Peak e were hetergenus and ranged in size frm 5 t 22 A; thse f Peak g had an average diameter f 8 ± 8 A. Studies by Mlecular Sieve Chrmatgraphy As shwn in Figure 6, the elutin prfile f HDL incubated with yielded tw majr peaks labeled and. Peak eluded befre, and peak after, the rignal HDL. The elutin time f the micelle peak almst cincided with that f Peak. With the increment f cncentratin, Peak exhibited a higher absrbance reading at 28 nm; at the cncentratin f f 5 t 1 mg/ml, it exceeded that f Peak. The cmpsitin f these peaks varied accrding t the amunt f in the system (Figure 6). n each case, Peak 1 cntained less than Peak. n the extreme case examined (1 mg ), abut 8% was present in the cmplex. When Peaks c and d frm the ultracentrifugal experiments shwn in Figure 3 were applied t this clumn, they eluted in the regins f Peaks and respectively. () (2) a b c d e f g a b c de f g Figure 4. Plyacrylamide gel electrphretic patterns f the ultracentrifugal peaks frm the experiments summarized in Figure 3. After dialysis against.5 M NH 4 HCO 3 buffer (ph 8.2), 1 ^\ f each sample was separated by SDS (1) r 8 M urea (2) plyacrylamide gel electrphresis.

7 424 ARTEROSCLEROSS VOL 4, N 4, JULY/AUGUST 1984 C Figure 5. Electrn micrgraphs f negatively stained preparatins f incubated mixtures f HDL- WR-1339 separated by density gradient ultracentrifugatin (see Figure 3). The varius fractins were dialzyed against 5 mm NH 4 HCO 3 (ph 8.) applied nt carbn film-cated cpper girds and negatively stained by a 2% slutin f Na phsphtungstate (ph 7.). A. HDL cntrl (Peak a). B. HDL plus 2 mg/ml (Peak b). C. HDL plus 1 mg/ml (Peak e). D. HDL plus 1 mg/ml (Peak g). Bar = 1 A. Effect n LDL Figure 7 shws the single spin ultracentrifugal prfile f an incubated mixture f and LDL (-1 mg -2 mg LDL prtein). At a cncentratin f 5 mg/ml, caused the appearance f tw majr peaks. Peak cntained, prtein, and lipids, whereas Peak cntained mainly. By gradient gel electrphresis, the incubated LDL- cmplex was increased in size (abut 2%) as cmpared t the cntrl LDL. By electrn micrscpy, untreated LDLs were mainly spherical, whereas the nes incubated with (2 mg /2 mg LDL prtein) appeared flattened with the tendency t frm ruleaux. At a higher cncentratin f (1 mg /2 mg LDL prtein), there was a marked decrease f ruleaux figures replaced by circular structures embedded in a unifrm backgrund f small D granular elements f an unknwn nature (Figure 8). The mean diameter f the cntrl particles was 243 ± 1 A and fr the defrmed particle 18 x 22 A; in the presence f 2 mg, the average diameter f the spherical particles was 285 ± 15 A and fr the flattened LDL, 33 ± 25 x 17 ± 25 A. n the presence f 1 mg, the spherical particles had a diameter f 241 ± 4 A. Effect f n LCAT n Vitr WR-1339 caused an inhibitin f LCAT. As shwn in Figure 9, the activity f the enzyme in plasma decreased as a functin f the cncentratin f the detergent. At a cncentratin f 1 and 2 mg /ml plasma, LCAT activity was less than 3% and 15% f the cntrl, respectively.

8 TRTON WR 1339 AND PLASMA LPOPROTENS Yamamt et al i- Tri tn mg / Tc B PL O.2- O.- C Trit ifc Pr Tc PL B _SS_ E c O CO C\J Pr Tc PL 9T9 TaDj O c 1 mg t.2- O.l- CX V Vi Fractin Number [ 1 u \ PT f til K Pr PL T< 1 15 U di i Figure 6. Sepharse 4B elutin prfile and chemical analysis f rhesus HDL after incubatin with varius cncentratins f fr 2 hurs at 37 C. The HDL preparatin cntained 3 mg prtein/ml. One mililiter f each sample was applied t a Sepharse 4B clumn and eluted with.5 M phsphate (ph 7.2) at a flw rate f 2 ml/hr at 6 C. The eluates were mnitred at 28 nm. V and Vi indicate the elutin vlumes f Blue Dextran 2 and L-tyrsine, respectively. The chemical cmpsitin (weight %) f Fractins and are shwn n the right. n Viv Studies Structural Changes f Lipprtein Attending the ntravenus njectin f WR-1339 Figures 1 and 11 shw the changes in the single spin prfile and distributin f idinated ap A-l, ap A-ll, and as well as the cncentratin f ap A-, ap B, and ap E befre and 15 minutes after the injectin f (6 mg/kg bdy weight) in the nrmchlesterlemic (N) mnkey and the hyperchlesterlemic (H) mnkey. n the N mnkey (Figure 1), the tw majr subsets f HDL (d = 1.8 g/ml, and d = 1.17 g/ml) shifted t a lighter density (d = 1.74 g/ml and d = 1.86 g/ml) and were increased in cncentratin as assessed by absrbance readings at 28 nm. Hwever, the 28 nm peak cmprised bth the HDL prtein and since when studied alne had a maximal absrbance at 278 nm. Mst f the radiactivity was detected in the lighter HDL. Mrever, 37% f ap A-l was fund in the bttm fractin and had the same specific activity as the ap A-l in

9 426 ARTEROSCLEROSS VOL 4, N 4, JULY/AUGUST j Omg Pr Tc 49 PL TC 24l5l OA 2 mg Tc 41 PL 2 c JQ V) mg mg T ' ' T Fractin Number Pr n L T n 1 29 Tc 37 PL 19 Pr Tc 74 6l 14 6l Pr Tc 34 PL TC PrTCPt M85J Figure 7. Single-spin density ultracentrifugal prfile and chemical analysis f incubated mixtures f LDL and. Preparatins f rhesus LDL (2 mg prtein/ ml) were incubated with varius cncentratins f (final cncentratin f ;,2,5 and 1 mg/ml) fr 2 hurs at 37 C. The samples were separated by single-spin density gradient ultracentrifugatin. After separatin, the effluents were cntinuusly mnitred at 28 nm and.4 ml fractins. The tw main peaks labeled and were analyzed fr chemical cmpsitin and electrn micrscpy. A B 6 Figure 8. Electrn micrgraphs f negatively stained preparatins f incubated mixtures f LDL and WR The samples were btained by density gradient ultracentrifugatin (see Figure 7). The samples were dialyzed against 5 mm NH4HCO3 buffer, (ph 8.) applied nt carbn film-cated cpper grids and negatively stained with a 2% slutin f Na phsphtungstate (ph 7.). A. LDL cntrl. B. LDL plus 2 mg/ml. C. LDL plus 1 mg/ml. Bar =1 A.

10 TRTON WR 1339 AND PLASMA LPOPROTENS Yamamt et al i.o 2. ( mg/ml whle plasma) Figure 9. n vitr effect f n LCAT activity f whle plasma. After preincubatin f whle plasma (1 J.\) with albumin- 3 H-labeled chlesterl emulsin (3 fi\) fr 4 hurs, varius cncentratin f (3 /nl) were added t plasma and the mixtures were incubated fr 2 hurs at 37 C. The reactin was started by adding 2 ^l f.1 M mercaptethanl slutin and incubating fr 1 hur at 37 C. The LCAT activity is given as the percentage f labeled chlesterl acylated per hur. HDL. n turn, radiactive ap A-ll was nly detected in the HDL fractin. Ap E, which in the cntrl sample was distributed between VLDL, intermediate density lipprtein (DL), and LDL, was ttally shifted t the bttm fractin after injectin. N change in density distributin and cncentratin was fund assciated with ap B. As shwn in Table 2, the chemical analysis f pled HDL frm the single spin fractins befre and after injectin in the N mnkey revealed a decrease in prtein, the presence f and n change in the lipid cmpnents. As assessed by gradient gel electrphresis (Figure 12), caused an increase in the size f the HDL particles. This finding was crrbrated by electrn micrscpy studies shwing that after the HDL particles were larger and mre hetergenus (befre a pled HDL sample had an average diameter f 95 ± 5 A and after 18 ± 4 A). Plyacrylamide gel electrphretic analyses in SDS and urea indicated a relative decrease f ap A-l cmpared t ap A-ll (data nt shwn). n the H mnkey (Figure 11), the single spin prfile befre shwed a single brad LDL and a single dense HDL peak befre injectin. After, the HDL peak (d = g/ml) shifted t a lighter density (d = 1.93 g/ml). The apprtein redistributin seen in the N mnkey was als seen with the H mnkey. The physicchemical changes in the prperties f HDL induced by were similar in the N and H mnkeys except that in the latter, the plasma cncentratins f ap B and ap E were higher. Prgressin and Regressin f Plasma Changes with a Single ntravenus Administratin f Aplipprteins, Lipids, and in Whle Plasma. As shwn in Figure 13, the single intravenus injectin f int either the N r the H mnkey caused a rapid fall f plasma ap A-l. This fall was particularly dramatic in the H mnkey, where the ap A-l was n lnger detected 1 day after the injectin and was still absent 3 days later. By the seventh day, there was partial restratin f the ap A-l levels; these were back t the pre- range after 14 days. Althugh the changes were less marked, ap A-l levels reacted similarly in the N mnkey. The changes in ap B and ap E were als mre prnunced in the H than in the N mnkey. Fr bth apprteins, the peak maximum was bserved 1 day after the injectin and returned t nrmal after 14 days. Elevatin f serum chlesterl and triglycerides was als bserved in the H mnkey shwing a marked inverse relatinship with the drp in serum Table 2. Physicchemlcal Parameters f HDL Befre and 15 Minutes After njectin f Nrmlipidemic mnkey Hyperiipidemic mnkey Befre 15 min after Befre 15 min after Diameter (A) Hydrated density (g/ml) Chemical cmpsitin (weight%) Prtein Ttal chlesterl Phsphlipid Triglyceride 9 1.8, , The analyses refer t a mixture f the tw HDL subsets. The size f the particles was btained by gradient gel electrphresis

11 428 ARTEROSCLEROSS VOL 4, N 4, JULY/AUGUST 1984 ap A-l levels. The plasma levels immediately after injectin int the N and H mnkeys were 1.46 and 1.39 mg/ml plasma, respectively. The levels f the detergent decreased at a much faster rate in the H mnkey than in the N mnkey. Overall, it tk 2 weeks befre the plasma was free f detergent and returned t nrmal. Electrphretic and Ultracentrifugal Changes. When pst- plasma was applied t 1 % agarse films (Figure 14), the a, band stained by Fat Red 7B disappeared within 15 minutes frm the injectin. After 1 day, the lipid-stainable band was in the B regin. After 1 week the a, band reappeared, and 2 weeks later the electrphretic prfile returned t its riginal pattern. Similar changes were bserved with the H mnkey except that after 1 day, a marked lipidstainable material was seen between the rigin and the (3 regin. After 2 weeks, the pattern returned t nrmal. This ttal reversibility as a functin f time was supprted by the single spin ultracentrifugal Absrbance -«(28nm) Befre injectin (N-MONKEY) 5 min after injectin (N-MONKEY) (cpm) *» ap E 4 (ug/itil) ap B (ugyipl) i Fractin Number Fractin Number H 1 i r Density ( g/ml) Density (g/ml) Figure 1. Alteratins in density gradient prfile and distributin f idinated ap A-l, ap A-ll, and, and the cncentratin f ap A-l, ap B, and ap E befre and 15 minutes after injectin f (6 mg/kg bdy weight) in the nrmchiesteriemic (N) mnkey. Plasma (1. ml) was applied t a single-step density gradient and spun t ispycnic equilibrium (66 hurs, 39, rpm, 15 C). The density prfiles were all recrded at 28 nm. Each tube was mnitred fr radiactivity. mmunquantiftcatin f apprteins was perfrmed n every ther tube. ^

12 TRTON WR 1339 AND PLASMA LPOPROTENS Yamamt et al. 429 data (Figure 15). The ultracentrifugal prfiles f the N mnkey shwed that the changes induced by affected the HDL peak and that the changes were already prminent 15 minutes after administratin and reverted t the pre- pattern after 2 weeks. As shwn by the data in Figure 1 and ur current studies in vitr, the bserved density changes were caused by the incrpratin f int the HDL particles and als by the displacement f ap E and ap A-l t the bttm fractin. n the H mnkey, the changes were mre dramatic; after 1 day there was a marked elevatin f the VLDL-DL peak, whereas there were n peaks in the areas crrespnding t LDL and HDL. After 14 days, the pattern was similar t that seen befre injectin. Clearance f dinated Ap A-l, A-ll and frm Plasma Befre and After njectin f WR-1339 The results f these studies are shwn in Figure 16. The disappearance curves f intravenusly injected idinated ap A-l, ap A-ll, and were biexpnential. After 24 hurs, abut 9% f the radiidinated ap A-l had disappeared frm the Absrbance (28nm) Befre injectin (H-MONKEY) 5 min after injectin (H-MONKEY) ap B (u/ul) ap A- (ug/tul) i-i Fractin Number Fractin Number ^ K) B Density ( g /ml) Density ( g/ml) Figure 11. Alteratins in density gradient prfile and distributin f idinated ap A-l, ap A-ll, and, and the cncentratin f ap A-l, ap E, and ap B befre and 15 mintues after injectin f in the hyperchlesterlemic (H) mnkey (See legend t Figure 1 fr technical details.)

13 43 ARTEROSCLEROSS VOL 4, N 4, JULY/AUGUST 1984 Figure 12. Plyacrylamide gradient gel electrphresis f pled HDL btained frm the single-spin ultracentrifugatin studies reprted in Figures 1 and 11.1 f±\ f HDL was applied t gradient gels PAA 4/3 and electrphretic separatin was carried ut at 14 C at 125 V fr 2 hurs, a = pled HDL f the hyperchlesterlemic (H) mnkey befre injectin (tubes 19-25); b = pled HDL f the H mnkey 15 minutes after injectin (tubes 16-22); c = pled HDL f the nrmchlesterlemic (N) mnkey befre injectin (tubes 16-24) and d = pled HDL f N mnkey 15 minutes after injectin (tubes 13-21). The average size f the particles was calculated frm the calibratin curve f reference prteins. Their radii were: thyrglbulin (85. A), apferritin (61. A), lactate dehydrgenase (4.8 A), and bvine serum albumin (35.5 A). a t b c d std Thyrglbdin Apferritin -Catalase -Lactati dehydrgenase -BSA j/ml) E E M _O a> c~ c a> i 1.U N-MONKEY ^ ^ ^ ^ ap A ap EN i apb / : -Ca, >.. Ttal Chlesterl Triglyceride H-MNKEY Ttal Chlesterl * " A.^ Triglyceride i 3 r Time ( days ) Figure 13. Time changes in the levels f plasma ap A-l,, ap B, ap E, ttal chlesterl, and triglycerides after a single intravenus injectin f (6 mg/kg) int the nrmchlesterlemic (N) and the hyperchlesterlemic (H) mnkey. i i i i i r 1 14

14 TRTON WR 1339 AND PLASMA LPOPROTENS Yamamt et al. 431 be fre 15 mm day 14 " (V Figure 14. Time changes in the agarse gel prfiles f the plasma f the nrmchlesterlemic mnkey befre and after the intravenus injectin f 6 mg /kg. Whle plasma (3 ^ was applied t the agarse gel; after electrphresis, staining was dne with Fat Red 7B. plasma f the H mnkey cmpared t abut 5% in the N mnkey (A). This was als the case with ap A- (B). The t 1 /2 value f radilabeled ap A-l frm the die-away curve during the first 27 minutes after the injectin was 13 hurs in the N mnkey and 6 hurs in the H mnkey. Fr ap A-ll, the tvfe value was 24 hurs fr N and 8 hurs fr H. Frm the slw phase f the curve f the specific activity values, the t 1 /2 f ap A-l was 3.8 days and that f ap A-ll, 4.8 days. Similar values were btained after. When radilabeled was injected in tracer amunts during the first day (C), its clearance was faster in the H mnkey than in the N mnkey. After 3 days, abut 9% f the injected had disappeared frm the plasma f the H mnkey as cmpared t 6% in the N mnkey. Between 3 days and 14 days, the slpes f the curves were cmparable, with an estimated t 1 /2 f 4.1 days. Discussin By cmbining ispyenic density gradient ultracentrifugatin, clumn chrmatgraphy, electrphresis, electrn micrscpy, immunchemical and radilabeling techniques, we shwed that when plasma f rhesus mnkeys interacts in vitr with the nninic detergent WR-1339 abve the critical micellar cncentratin (cmc) f this detergent (unpublished data), changes in bth LDL and HDL particles ccurred. Hwever, within the cncentratin ranges f used, the effects n HDL were mre marked and began t ccur at lw detergent levels (1-2 mg /mg HDL prtein). At these levels, there was a prgressive replacement f ap A-l and ap E by the detergent at the HDL surface withut a detectable effect n the lipids r the ther aplipprteins, particularly ap A-ll. Even at higher cncentratins f, this apprtein was mre resistant t displacement by than ap A-l, an bservatin which is in keeping with the previusly reprted 33 " 35 differences in affinity f these tw aplipprteins at the HDL surface. The mass f added t the HDL particles caused an increase in their particle size until the detergent mass reached a level at which the lipids separated int the detergent micellar phase frm the prtein miety. Thus, depending n cncentratin, culd act either as a stabilizer f the lipprtein particle by substituting fr ap A-l at its surface r cause disruptin by cmpeting fr the lipids nrmally interacting with the apprteins. At this stage, mixed micelles f detergent and lipids were frmed which culd absrb ap A-l at their surface, generating a lipid-apprtein cmplex. Hwever, nt all the ap A-l displaced frm the HDL surface fllwed this fate; sme may have assciated with detergent mnmers as described by Reynlds and Simn 36 fr the interactin f human ap A-l and A-ll with the aninic detergent sdium ddecylsulfate and as Makin et al. 37 described catinic detergent tetradecyltrimethyl ammnium ins. The displacement f ap A-l frm the HDL surface by may partially accunt fr the results f ur LCAT studies, which shwed an inverse relatinship between enzymatic activity and the amunt f ap A-l displaced frm the surface in keeping with previus studies by this labratry 38 and in viv studies in the rat. 39 The in viv studies als shwed a great affinity f WR-1339 fr the HDL particles. The single' intravenus injectin f the detergent int either a nrmlipidemic rhesus mnkey r a mnkey which was made hyperchlesterlemic by an athergenic diet was fllwed by imprtant structural changes in these lipprteins, which were dependent upn the levels f in the plasma. These in viv changes were bth quantitatively and qualitatively similar t thse elicited by the detergent when incubated in vitr with whle plasma, at least within the limits f the initial plasma levels f prduced by the injectin f 6 mg/kg. We avided injecting intravenus dses f the detergent abve 6 mg/kg t avid red bld cell hemlysis. Scanu et al.' 2 ' 13 reprted that in the dg, has a hemlytic actin n canine red bld cells in vitr and in viv. As t the preferential effect f WR-1339 n HDL, Prtman et al. 11 reprted an immediate (after 5 minutes) 7% drp in HDL plasma levels f d = 1.63 t 1.21 in squirrel mnkeys injected intravenusly with. Mrever, a disruptive actin f

15 432 ARTEROSCLEROSS VOL 4, N 4, JULY/AUGUST 1984 WR-1339 n HDL in the rat was reprted by shikawa and Fidge 15 after a single intravenus injectin f (25 mg/kg). As in ur studies, the mst striking bichemical lesin induced by was the dissciatin f ap A-l frm the HDL particles, the extent f which was dependent n the plasma cncentratins f. Since ap A-l dissciatin als ccurs in the dg (unpublished bservatins), it is prbable that such a dissciatin may have ccurred in all f the previus studies where was used in dses between 3 mg/kg and 5 mg/kg/bdy weight t prduce a hyperlipidemic state. 2 ' * 5> 1> 13 ' 15 Based n calculatins f the rati between bld vlume and bdy weight, these dses crrespnd t a detergent level f 8 t 13 mg/ml f plasma, which ur results indicate wuld cause the ttal displacement f ap A-l frm HDL. The interactin f with HDL had imprtant metablic cnsequences indicated by the rapid drp f plasma HDL and the rapid clearance f ap A-l and ap A-11 frm the circulatin. Several factrs may have cntributed t these events. Pssibly nce the HDL particles became cated by, they culd n lnger interact physilgically with ther plasma lipprtein systems, enzymes, r membrane receptrs 16 and s they underwent cellular uptake by nnspecific mechanisms. The invlvement f the reticul-endthelial system in dgs receiving WR-1339 has been dcumented. 13 n thse studies, a marked accumulatin f lipid-laden macrphages in the spleen, liver, lymph ndes, and arteries was bserved after the chrnic administratin f. Still used electrn micrscpy t demnstrate 4 the presence f -lipid cmplexes in the endthelium f the arterial intima in rats and rabbits given several intravenus injectins f WR n ur studies, the clearances f ap A-l and ap A-11 were mre rapid in the animal that was n an athergenic diet N-MONKEY TME H-MONKEY btfrt <\J 111 u z < tc </) CD 4 Smln 3hr«day 3 day* 7 day* 14 days L6 1.1 L15 L3 L4 LOS 1.1 DENSTY ( g/ml) DENSTY ( g/ml ) Figure 15. Time changes f the plasma lipprtein distributin after a single intravenus injectin f (6 mg/kg) int nrm- and hyperchlesterlemic mnkeys. One mililiter f plasma was separated by single-spin density gradient ultracentrifugatin and the eluates frm each tube were cntinuusly recrded at 28 nm.

16 TRTON WR 1339 AND PLASMA LPOPROTENS Yamamt et al. 433 and we speculate that this diet acted as an activatr fr the reticul-endthelial system, favring the phagcytic prcess f the -cntaining HDL. This interpretatin is in keeping with the bservatin f a rapid plasma clearance f radilabeled in the hyperchlesterlemic mnkey. Mrever, a structur- O rr a> [ l8l l] ap A- k \ ^ ^ \ \\ V \ a a A A l] ap A- ~ ^ i] 1 N N H H --. " T befre after befre after ^3^~- = 4.8da Time (days) it ii days Figure 16. Curves f plasma disappearance f ap A-, 12S l-ap A-ll, and 125 l- befre and after a single intravenus injectin f (6 mg/kg) int nrm- and hyperchlesterlemic mnkeys. The radiidinated aplipprteins (each 2 /xci) were injected intravenusly at the same time int bth mnkeys and bld specimens were taken at time intervals. One mnth later, the same experiment was repeated except that (6 mg/kg) was injected 3 minutes after radiidinated ap A- and ap A-ll were injected intravenusly. The plasma disappearance f 12S l- was determined after intravenus injectin f 2 fic\ f the radiactive detergent (6 mg/kg). The experiment was carried ut apprximately 3 mnths after the turnver studies with radiactive ap A- and ap A-ll. 14 al alteratin f HDL may accunt fr the hyperlipidemia fllwing the intravenus injectin f. An accumulatin f VLDL in animals treated with WR-1339 has been nted in dgs, 13 rats, 15 and squirrel mnkeys 11 and has been attributed t abnrmalities in VLDL-LDL cnversin secndary t changes in HDL structure. 15 The displacement f ap E frm the HDL particles may als have cntributed t the alteratin in HDL catablism. shikawa and Fidge 15 shwed an abnrmality in ap C-ll metablism in the rat after administratin. At higher cncentratins, the -induced metablic abnrmalities becme mre cmplex because f the ptential invlvement f the liplytic enzymes 41 and ther plasma lipprteins. This wuld be true in animals subjected t repeated injectins f. We reasn that nce HDLs are n lnger present in plasma as intact particles, may assciate with the ther plasma lipprteins; and that nce thse sites are saturated, prduces structural changes at the cell membrane level. The structural changes f LDL induced by were small cmpared with thse bserved with HDL. This was prbably due t the structural characteristics f LDL and the fact that less than 1% f the added assciated with LDL. did increase the size and the hydrated density f LDL, but even with the high cncentratins f detergent used (1 mg/2 mg f LDL prtein), there was n displacement f ap B r lipid frm the lipprtein particle. n early studies n dgs, Scanu and Oriente 12 demnstrated that with a cncentratin f 4 mg f / mg LDL prtein, sme disruptin f LDL ccurs. Hwever, these are exceptinally high dses which can prduce hemlysis. Many years ag, Hirsch and Kellner 42 suggested that the actin f WR-1339 was cnfined t the bld alne and that it smehw "cated" fat particles, thus altering their physilgic and metablic states. Based n past and current studies in this labratry 12 ' 13 and studies by shikawa and Fidge, 15 we can state that the early lesin is cnfined t the HDL class, which is knwn t play several functinal rles in lipid metablism. 18 n this cntext, WR-1339 appears t be a very useful prbe fr studying the structure and functin f HDL bth in nrm- and hyperlipidemic animals. Acknwledgments The authrs thank Laura Harris and Tim Bridenstine frm the SCOR Athersclersis Cre fr helping in the bld samplings f the animals. Rbert Byrne and Celina Edelstein prvided useful cmments during these studies. Rse Sctt's wrk in manuscript preparatin was invaluable. References 1. Scanu AM. Factrs affecting lipprtein metablism. Adv Upid Res 1965;3: Hirsch RL, Kellner A. The pathgenesis f hypertipldemla induced by means f surface-active agents.. ncreased ttal bdy chlesterl in mice given WR J Exp Med 1956;14:1-14

17 434 ARTEROSCLEROSS VOL 4, N 4, JULY/AUGUST Kellner A, Crrell JW, Ladd AT. Sustained hyperlipidemia induced in rabbits by means f intravenusly injected surface-active agents. J Exp Med 1951;93: Fiser RH, Dennistn JC, Rimdsig RB, Beisel WR. Triglyceride secretin rates: Use f WR 1339 in the rhesus mnkey. J Nutritin 1974; 14: Otway S, Rbinsn DS. The use f nninic detergent ( WR-1339) t determine rates f triglyceride entry int the circulatin f the rat under different cnditins. J Physil 1967;196: Schurr PE, Schulz JR, Parkinsn TM. -induced hyperlipidemia in rats as an animal mdel fr screening hyperlipidemic drugs. Lipid 1972;7: Zilversmit DB, Hughes LB, Remingtn M. Hyplipidemic effect f pregnancy in the rabbit. J Lipid Res 1974; 13: Klauda HC, Zilversmit DB. nflux f chlesterl int plasma in rabbits with fasting hyperbetalipprteinemia. J Lipid Res 1974;15: llingwrth DR. Whipple LE, Prtman OW. Metablism f lipprteins in nn-human primates: Reduced secretin f very lw density lipprteins in squirrel mnkeys with dietinduced hyperchlesterlemia. Athersclersis 1975;22: llingwrth DR. Metablism f lipprteins in nnhuman primates. Studies n the rigin f lw density lipprtein apprtein in the plasma f the squirrel mnkey. Bichim Biphys Acta 1975;388: Prtman OW, Alexander M, Tanaka M, llingwrth DR. Triacylglycerl and very lw density lipprtein secretin with plasma f squirrel mnkeys. Bichim Biphys Acta 1977; 486:47-^ Scanu AM, Oriente P. hyperlipidemia in dgs.. n vitr effects f the detergent n serum lipprteins and chylmicrns. J Exp Med 1961 ;113: Scanu AM, Oriente P, Szajewski JM, McCrmack LJ, Page H. hyperlipidemia in dgs.. Athersclersis, diffuse lipidsis, and depletin f fat stres prduced by prlnged administratin f the nn-inic surface active agent. J Exp Med 1961 ;114: Friedman M, Byers SP. The mechanism respnsible fr the hyperchlesterlemia induced by WR J Exp Med 1953:97: shikawa T, Fidge N. Changes in the cncentratin f plasma lipprteins and apprteins fllwing administratin f WR-1339 t rats. J Lipid Res 1979;2: Scanu AM, Byrne R, Mihvilvic M. Functinal rle f plasma high density lipprteins. Crit Rev Bichem 1982; 13: Fless G, Wissler RW, Scanu AM. Study f abnrmal plasma lw-density lipprteins in rhesus mnkeys with diet-induced hyperlipidemia. Bichemistry 1976;15: Scanu AM, Edelstein C, Vitell L, Jnes R, Wissler RW. The serum high density lipprteins f Macacus rhesus. slatin, cmpsitin, and prperties. J Bil Chem 1973; 248: Nilssn J, Mannickarttu V, Edelstein C, Scanu AM. An imprved detectin system applied t the study f serum lipprteins after single step density gradient ultracentrifugatin. Anal Bichem 1981 ;11: Edelstein C, Lim CT, Scanu AM. The serum high density lipprtein f Macacus rhesus. slatin, purificatin, and characterizatin f their tw majr plypeptides. J Bil Chem 1973;248: Karlin J, Juhn D, Fless G, Scanu AM, Rubenstein AH. Measurement f rhesus mnkey (Macaca Mulatta) aplipprtein B in serum by radiimmunassay: Cmparisn f immunreactivities f rhesus and human lw density lipprteins. J Lipid Res 1978;19: Pfaffinger D, Edelstein C, Scanu AM. Rapid islatin f aplipprtein E frm human plasma very lw density lipprteins by mlecular sieve high perfrmance liquid chrmatgraphy. J Lipid Res 1983;24: Laurell CB. Quantitative estimatin f prteins by electrphresis in agarse gel cntaining antibdies. Anal Bichem 1976;15: Schnfeld G, Chen J-S, McDnnel WF, Jeng. Aplipprtein A-ll cntent f human plasma high density lipprteins measured by radiimmunassay. J Lipid Res 1977;18: McFarlane AS. Efficient trace-labelling f prteins with idine. Nature 1958; 182: Lwry OH, Rsebrugh NJ, Farr AL, Randall RJ. Prtein measurement with the Flin-phenl reagent. J Bil Chem 1951; 193: Allain CC, Pn LC, Chan CS, Richmnd W, Fu PC. Enzymatic determinatin f ttal serum chlesterl. Clin Chem 1974;2: Gall LL, Atasy R, Vahuny GV, Treadwell CR. Enzymatic assay fr chlesteryl ester hydrlase activity. J Lipid Res 1978;19: Bartlett GR. Phsphrus assay in clumn chrmatgraphy. J Bil Chem 1959;234: Kane JP. A rapid electrphretic technique fr identificatin f subunit species f apprteins in serum lipprtein. Anal Bichem 1973;53: Weber K, Osbrn H. The reliability f mlecular weight determinatin by ddecyl sulfate-plyacrylamide gel electrphresis. J Bil Chem 1969;244: Stkke KT, Nrum KR. Determinatin f lecithiitchlesterl acyltransferase in human bld plasma. Scand J Clin Lab nvest 1971;27: Lagcki PA, Scanu AM. n vitr mdulatin f the apprtein cmpsitin f high density lipprteins: displacement f aplipprtein A-l frm HDL by aplipprtein A-ll. J Bil Chem 1982;255: Rsseneu M, Setewey F, Middelhff G, Peeters H, Brwn WV. Studies f the lipid binding characteristics f the apprteins frm human high density lipprtein. Calrimetry f the binding f ap A-l and ap A-ll with phsphlipids. Bichim Biphys Acta 1976;441: Edelstein C, Halari M, Scanu AM. On the mechanism f the displacement f aplipprtein A-l by aplipprtein A-ll frm the high density lipprtein surface. Effect f cncentratin and mlecular frms f aplipprtein A-ll. J Bil Chem 1982;257: Reynlds JA, Simn RH. The interactin f plypeptide cmpnents f human high density lipprtein with sdium ddecyl sulfate. J Bil Chem 1974;249: Makin S, Tanfrd C, Reynlds JA. The interactin f plypeptide cmpnents f human high density serum lipprtein with detergents. J Bil Chem 1974 ; Scanu AM, Lagcki P, Chung J. Effect f aplipprtein A-ll n the structure f high density lipprteins: relatinship t the activity f lecithin-chlesterl acyltransferase in vitr. Ann NY Acad Sci 198;348: Sler-Argilaga C, Russell RL, Heimberg M. Effect f WR-1339 n lecithin-chlesterl acyltransferase in rat. Arch Bichem Biphys 1977;178: Still WJS. The affect f n the arterial endthelium: a scanning and transmissin electrn micrscpy study. Pari Arterielle 1974;2: Schtz MC, Scanu AM, Page H. Effect f WR-1339 n lipprtein lipase f rat plasma. Am J Physil 1957;188: Hirsch RL, Kellner A. The pathgenesis f hyperlipidemia induced by means f surface active agents.. Failure f exchange f chlesterl between the plasma and the liver f rabbits given WR J Exp Med 1958;14:4-14 ndex Terms: WR 1339 plasma lipprteins HDL structure ap A-l ap A-ll rhesus mnkeys