170

nim.h~mlc, et niophysi~'a A,~a. 1082(19911170-176 a: 1991 ElsevierSciencePublishersB.V.0O05-2760/91/$03.50 A D O N I S 0O05276091O011SD

Isolation and characterization of human Lp-B lipoprotein containing apolipoprotein B as the sole apolipoprotein J e a n M a r i e B a r d ~, L a u r e n t C,indeliex 1, Q e n e v i 6 v e A g n a n i 1, V 6 r o n i q u e C l a y e y 1, G ~ r a r d T o r p i e r 1, A r m i n S t e i n m e t z 2 a n d J e a n C h a r l e s F r u c h a r t ! Serlit~ I N S E R M U. 325, lnsttmt Pasfeur, Lille (France) z Universit~'t Marburg, Zentrura Innere Medizin End~rinolog& und St~ffwechsel. Marburg (FR.G.)

(Received 30 July 199O) Key words: Lipoproteinparticle; ApolipoproteinB; lmmunoaffinitychromatography A sequential immunoalfinity chromatography procedure was developed to isolate from whole normolipidemic human plasma a subpopulation of apoB containing particles (Lp-B) which is virtually free of non apoB protein. The absence of non apoB protein in Lp-B was assessed by enzyme immunoassay against apolipeproteies A-I, A-II, A-IV, E, C-III and fa). Electron microscopy and fractionation of the isolated parades by gel filtration demonstrated that these partleles ~rere heterogeneous in size. However, most of them had diameters between 18 and 26 nm. These particles were found to be rich in cholesterol (molar ratio cholesterol/apoB = 2246 4- 995) poor in triacylglycerol (molar ratio wincylglycerol/ almB = 555 + 518) and had a phespboliplds/apoB molar ratio of "/13 + 348. Most of the cholesterol was estetifled (66% + 5%). Lp-B particles bound to the apoB, E receptor of HeLa cells with a lower alfinity than LDL prepared by altraeentrifugation (L030 k g / l < d < 1.053 k g / I ) . (K n - 18.9 vs 10.5 nmol/I). Introduction Results of immunological characterization and apolipoprotein quantification of the major lipoprotein density classes have shown that each consists of several qualitatively distinct lipoprotein particles rather than single homogeneous lipid-protein complexes [1]. In other words, lipoproteins appear to be a mixture of lipoprotein particles characterized by similar hydrated densities or electrical charges but differing in apalipoprotein composition. A previous study [2] showed that immunoaffinity chromatography represents the method of choice for the isolation of lipoprotein particles defined by their apolipoprotein constituents. This approach is considered to have little, if any effect on the structural integrity of the particles [2] and in particular seems to avoid the dissociation of apolipoprotein E from lipoproteins as it is observed during standard ultraccotrifugation [3]. Recently a method for the isolation of Lp-B from L D L has been described [4]. This method does not avoid ultraecntrifugation and does not allow the preparation of Lp-B directly from whole plasma. In the

Correspondent: J.M. Bard,Serlia,lnstitut Pasteur, L rue du profes ~ur Calmette, 59O19LilleC&lex{France}.

present paper we describe the isolation from normolipidemie human plasma of lipoprotein particles containing only apofipoprotein B using sequential immunonffinity chromatography with antibodies against apollpoprotein A-I, A-ll, A-IV, B, E, C-III and (a). Mates~als and Methods Preparation of immunosorbents

Pure apolipoproteins and LDL were obtained as previously described and animals were immunized to raise monoclonal antibodies [5] against apoA-! [6], apoA-II [6], apoE |6] and apo(a) [5] and polyclonal antibodies [7] against apoA-IV [8], apoB [9l and dpoC-

nl 161.

Antibodies specific to either apoA-I, apoA-II, apoAIV, apoB, apoE, apoC-lll or apo{a) were isolated from the respective antisera by affinity chromatography. LDL, apoA-l, apoA-IL apoA-iv, apoE, apoC-lll and Lp(a) were cova]ently coupled to CNBT activated Sep. harose 4B according to the procedure of the manufacturer (Pharmacia Fine Chemicals Uppsala, Sweden).The specific anti-apolipoprot¢in A-I, A-II, A-IV, B. E, C-Ill and (a) IgG were isolatec~ by incubation of total IgO obtained by precipitation from antisera using ~7~ saturated sodium sulfate with the corresponding ira-

munosorbers. Elution of the specific lgG from the antigen gel was accomplished using 0.2 M glycine, pH 2.8. The antibodies employed in this study were previously documented 1o be specific for either apolipoproteins A-l, A-II, A-IV, B. E. C-Ill or Lp(a) by Ouchterlony gel diffusion and specific non competitive enzyme immunoassays (ELISA) [101. Affinity-isolated antibodies to these apolipoproteins were covalently coupled to CNBr activated Sepharose 4B according to the procedure of the manufacturer.

Lipid analysis Total cholesterol (TC), free cholesterol (FC), triacylglycerol (TG) and phospholipids (P) were measured by specific enzymatic test kits from Boehringer-Mannheim. Mannheim (F.R.G.) Esterified Cholesterol (EC) was estimated as the difference between TC and FC.

Isolation of lipoprotein particles by immunoaffinity chromatography Blood samples were obtained from seven normolipidemic (cholesterol < 6.50 retool/l, triacylglycerols < 1.6 retool/I) subjects in the morning after an overnight fasting period, Venous blood was drawn into vacutainer tubes containing disodium EDTA (50 pM). After separation of plasma by low-speed (3000 × g) centrifugation at 4oC, 0.6 mM chloramphenieol, 1.6 mM glutzthione, 10 mM caproic acid, 1 mM EDTA and 10 mM D-phenylalanyl-t-prolyl-t.-arginine chloromethyl ketone (PPACK) were promptly added. The plasma was immediately used for isolation of lipoproteins by immunoaffinity chromatography. Plasmas were subjected to immunoaffinity chromatography at 20°C and monitored at 280 nm. The lipoprotein fraction named Lp-B was obtained according to the scheme presented in (Fig. l). Complex apoB containing particles (L2-B (cx)). 40 ml of fresh human plasma were collected after overnight fasting. The plasma was diluted to 200 ml ~vitb buffer containing 0.01 M Tris-HCl, 0.15 M NaCI, 0.3 mM EDTA, 1.5 mM NaN3 and 10 I~M phenylmeti~ylsnlfonyl fluoride (PMSF) p H 8 and passed through the anti apoB affinity column at a flow rate of 60 ml/h. Lipoproteins not bound to the immunosorbent were washed off with the buffer. Non specifically bound proteins, due to weak hydrophobic or ionic interaction, were further removed by washing the gel with the same buffer containing 0.5 M NaCI. Absence of apoB in these fractions was tested by ELISA [10]. The complex apoB-containing particles were elated with 3 M sodium thioeyanate (50 ml). A column containing 100 ml of Sephadex G25 was linked to the immunosorbent column, in order to separate immediately the lipoproteins from me dissociating agent (sodium thioeyanate). Two fractions were obtained, the first containing lipopro-

Plasma

tp B I t . )

I Lp BlCx) ~ t h o u t apo E

Lp 8{cx) without a

o

an

wn

OUI aN

CIII

Lp a Fig. I. Flow diagram ,*or immunoatfinRy column chromatography preparation of pure Lp-K r; r¢lalned fraction, ur;unte|alned ftacllon, • he buxc~ represent affinitycolumn~ containing monc~sp'~citicanti-

body di~(ed against abe apolipoprutein indicated. Pl~ma: starung whole plasma Lp-B(on): complex apoB containing particles elated from an anti B column. Lp-B (cx) wahoul apoE: complex apoB containing particles cluted from an anti D column, from which apoE conlaining parlielus have been re~vcd. L p - B (cx) without a p o E and apoC-[ll; complex apoB conttlnlng particles dutcd flora ,an anti B column, from which apoE and apoC-Ill containing particles have b~n removed. Lp-B:lipoproleins comainingapolipruteln B but {r~ of apoE. apoC-l[t and ape(a). teins but devoid of sodium thiocyanale and the second containing sodium thiocyanate as the principal constituent.

Lipoprowins comatning apoB but free of apoE, apoC II1 amlapo(a]. Comple× apoB containing particles eluted from the anti apoB column were incubated with anti apoE immunosorbent. The unbound and nonspecifically bound lipoproteins~ free Of apoE, were washed from the immunosorbent as described above. These unretained particles were then incubated with anti apoC-lll immunosorbent. The unbound and nonspeeifitally bound lipoproteins, free of apoC-Ill, were again removed from the immunosorbent as described above. These unretained particles were incubated with anti apo(a) immunosorbcnt. The unbound and nonspecifically bound lipoproteins, free of apo(a), were obtained from the immunosorbent as described above and named Lp-B. If necessary, trace amounts of apoA-L apoA-I1 or apoA-IV, were removed by chromatography over the corresponding immunosorbems according to the protocol described above. Lipoprotein particles were concentrated using YMI00 A micon membranes.

Apolipoprofein analysis Protein content of the isolated Lp-B was measured by the method of Lowry [ll I.

172 Absence of apolipoproteins (a), A-L A-IL E, C-III was checked by specific non competitive enzyme immunoassays (ELISA) [7,9]. Apollpoprotein B was quantified by ELISA [9]. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [12] was performed in discontinuous gel: 3% polyacrylamide stacking gel and 10-30% gradient gel. in order to check the absence of non apoB protein and the integrity of apolipoprotein B. Western blot [13] was used to identify the proteins present on the polyacrylamide gel. Analysis o/particle size The molecular size distribution of Lp-B was determined by gel filtration over a column of Superose 6 HR I 0 / 3 0 (Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated in 0.0l M Tris-HCL 0.15 M NaCI, 3. 10 -a M EDTA. 1.5-10 -3 M NaN 3, 2 . 1 0 ~ M PMSF (pH 8). The sample (200 /d) was duted at a constant flow rate of 12 ml/h. The absorbance of the eluent was monitored continuously at 280 nm. Human V LDL ( d < 1.006 kg/l) IDL (1.006 kg/I < d < 1.019 kg/l), LDL (1.019 kg/I < d < 1.063 kg/I) and H D L (t.063 kg/I < d < 1.21 kg/I) were used for calibration purposes. For electron microscopy the concentrated Lp-B were diluted with glass distilled water to give final concentration of about 0.2 to 0.4 mg/ml. After negative staining with 1% sodium phosphotungstate, pH 7.0 specimens were examined with a Philips EM 420 electron microscope. The mierosc,pe magnification was accurately calibrated using catalase crystals. Data corresponding to the counting of at least 500 particles were analyzed.

receptor mediated pathway 17]. Binding studies of b 1 labelled • or 12~Ialabelled lipoprotein particles LDL performed at 4°C on nearly confluent cells as described by Goldstein et al. [18] for LDL, gave a measure of cell surface receptor binding of the lipoproteins. These assays were performed in triplicate. Nonspecific bindipg was obtained by adding unlabelled LDL in excess (up to 20-fold) to the binding assay system. Results were expressed as the molar concentration of particles bound by mg cell protein, assuming that one lipoprotein particle containing one molecule of apolipop'otein B of molecular mass 549000 Da [19]. Kd was calculated by the method of Scatchard [20]. Results lmmunoaffinity chromatography The apoB content of Lp-B was determined by ELISA method. The absence of non apolipoprotein B proteins in Lp-B was checked by enzyme immunoassay. No significant trace of apoE, apoC-lll and apo(a) was found in Lp-B (less than 1%.on a molar basis), but in most cases, a chromatography over an anti apoA-l, apoA-II and apoA-IV immunosorbent was necessary to eliminate these apolipoproteins. SDS-PAGE and Western Blot confirmed the integrity of apolipoprotein B and revealed that apoB was the sole apolipoprotein in Lp-B. As shown in Fig. 2, a protein eomigrated with bovine serum albumin. Western blot confirmed that this band was definitely al-

Cell culture Lipoprotein deficient serum (LPDS) (fraction density > 1.21 kg/I), was prepared from a pool of normal human serum by two successive ultraeentrifugation steps [14]. HeLa cells were grown in Dulbeceo's minimum Eagle medium (DMEM) containing 10% (v/v) of fetal calf serum. 48 h before experiments, cells were preincubated in medium containing 10% (v/v) LPDS. All incubations were performed at 37°C in an atmosphere of 5% CO 2 in air. lodbtaHon LDL isolated by sequential ultracentrifugation in the density range 1.030 1.053 kg/l and lipoprotein partic!e~ were. radiolabelled with Na1251(Amersham) by the iodi,le monoehhiride method according to MacFarlane et al. [15], as modified for lipoproteins by Bilheimer et aL [16]. The specific radioactivity averaged 300 e p m / n g particle protein. Monolayer binding studies HeLa cells were used because they were previously shown to specifically bind. internalize and degrade LDL

94OOO 67 000 45 OC~O

3O 000 20 100 ~ 14 400 ~ ~.

Fig. 2. SDS-PAGE analysis of pure Lp-B, On the lefl lane. the following molecular weight eallbralion proleins we~ run: phosphoryb~e b 194000).bovh:e serum albumin (67000),ovalbumin (43000), carbonic ~hydras¢ 1300O0).soybean trypsin inhibitor (20t00), alpha laclalbumin (14400).On the right lane, pure Lp-Bwas run.

173 12o ! ,oo eo

20 0

7

a

9

10 15 12 13 14 15 I~

17

.~ ~OmB ml

Fig. 3. A char:leleristi¢ elulion profile of Lp-B obtained by gel filtralion chromalography by FPLC system using Superose 6. In ibis system. VLDL (d < 1.006 kS/I) are elated between ~ ml and 9.7 mL IDL (1.006 kg/I < d < 1.019 ks/I) b e t w ~ 9.7 ml and 11.5 mL LDI. (1.019 kg/I < d < 1.063 kg/I) between 11.5 ml and 15.5 *hi and HDL ll.063 hg/I < d < 1.21 kg/ll between 16 ml and 18.5 mL

b u m i n , As estimated by the difference between the p r o t e i n c o n t e n t d e t e r m i n e d by the L o w r y m e t h o d a n d the a p o B c o n t e n t d e t e r m i n e d by E L I S A , the m o l a r ratio b e t w e e n a l b u m i n a n d a p o B , i.e., the n u m b e r of a l b u m i n molecules p e r particles, was a r o u n d one.

Chemical analysis T a b l e I presents the lipid c o m p o s i t i o n of the differe n t isolated L p - B particles. If o n e assumes t h a t there is o n e molecule o f a p o B (molecular mass 549000 D a ) p e r particle, the m e a n n u m b e r of cholesterol molecules cartied by each L p - B particle was 2246 + 995. Cholesterol esters d o m i n a t e d in this fraction f66% 4- 5%). T h e n u m b e r o f triacylglycerol molecules p e r particle ranged bet w e e n 160 a n d 1660 ( m e a n = 555 4- 518). Phospholipids r a n g e d b e t w e e n 420 a n d 1470 ( m e a n - 713 4- 348).

Analysis of Lp-B size L p - B was fractionated by F P L C u s i n g Superose 6 ( P h a r m a c i a Fine Chemicals. Uppsala. Sweden). This gel filtration c h r o m a t o g r a p h y is reproducible a n d allows c o m p a r i s o n between different experiments. Each ehition profile d e m o n s t r a t e d that Lp-B particles were heterogeneous in size (Fig. 3). A l t h o u g h 0 - 5 ~ of the particles eluted w i t h V L D L (clution volume 8 - 9 . 7 m l ) a n d 1 0 - 1 5 % w i t h I D L (elution v o l u m e 9.7-11.5 mi), most particles ( 7 5 - 9 0 % ) eluted in the L D L size r a n g e (clution volutr, e l l . 5 - 1 5 . 5 ml). A few particles (0 10%) even eluted in the H D L size r a n g e (elutiort v o l u m e snore t h a n 15.5 ml). I n s o m e e x p e r i m e n t s ( n ~ 3). lipid c o m p o s i t i o n was determined in L p - B particles isolated f r o m V L D L ( d < 1.006 k s / I ) a n d L p - B particles isolated f r o m L D L (1.030 < d < 1.053 k s / I ) . L p - B particles isolated flora V L D L were m u c h richer in triacylglycerol (5317 + 2080 vs. 248 + 39) t h a n particles isolated f r o m L D L Cholesterol (4490+_ 1813 vs. 2482 _+ 17) a n d p b o s p h o lipids (2637 + 883 vs. 584 + 256) ~vere also f o u n d increased in L p - B particles isolated f r o m V L D L w h e n c o m p a r e d with particles isolated f r o m L D L Cholesterol was eslerified to a lesser extent in L p - B particles isolated f r o m V L D L t h a n in particles isolated f r o m L D L (cholesterol e s t e r s / t o t a l c h o l e s t e r o l - 0.45 + 0 . 1 4 vs. 0.67 + 0.03). Electron microscopy of t h e w h o l e Lp-B, large L p - B (eluted between 8 ml to 11.5 m l ) a n d small Lp-B (eluted b e t w e e n 11.5 ml to 15.5 m l ) revealed spherical particles. T h e size distribution of these different fractions calculated by electron microscopy (see Fig. 4) c o n f i r m e d the size heterogeneity of Lp-B. Fig. 5 represents h i s t o g r a m plots of the size distribution of Lp-l~ particles. T h e particle size r a n g e of the m a j o r subspecies in L a r g e Lp-B a n d Small Lp-B was respectively 2 6 - 3 0 n m a n d 18-22 nm.

TABLE 1

Molar c~positl~ o1"Lp-B Results are given as molar ratios considering 388. 877 and 775 as Ih¢ mean molecular weight for chol~t¢~], lriacylgiy~ol and phospholipids respectivdy. TC/B: total chol~lerol/apoB. CE/B: ehnleslerol ~SlC~/apoB. TG/g: tnacy]giyccrol/apoB. P/B: phospho0pids/apoB. TG/TC: triacylglycerol/total cholesterol. CE/TC: cholesterol esters/tolal cholesterol. P/TC: ph~pholipids/total cholesterol Eapefiments 1 2 3 4 5 6 7

TC/B 1237 4112 1461 2488 2635 1517 2255

CE/B 718 282 1016 1697 1583 1088 1537

TG/B 412 588 160 261 1660 20,1 507

P/B 418 I a68 668 533 669 532 703

mean standard deviation

2246 995

1495 685

555 518

713 348

TG/TC 033 0 14 0 11 a I1 o.63 0.13 0.26

CE/TC 0.58 0.68 0.70 0.68 0.71 0.72 0.68

p/TC 0.34 0.36 0.46 o.21 0.30 0.38 0.3l

0.25 0.19

0.66 0.05

0.33 0.08

r

174

//

lished [4], the use of sequential chromatography avoids the ultracentrifugation step and allows the purification of Lp-B from whole plasma. This approach is a commonly used fra¢tionation procedure and is considered to have little, if any, effect on the structural integrity of lipoprotein particles [2]. In particular it avoids dissociation of apolipoprotein E from the lipoprotein particles as it is observed using standard ultracentrifugation procedures [31. The presence of albumin in significant %

l/

5O

Fig, 4. Negativelystained electron micrograph of Lp-B (pattern a). Small Lp 0 (clution votume 11.5 15.5 ml} (pattern b) and Large Lp-B {elution volume 8 11.5 ml). (pattern e). The enlargement is 92000401d,

0 < 1 0 1 0 14 18 22 25 30 34 38 42 46 50 5 4 > 5 4

Binding of Lp-B to HeLa cells Lp-B was investigated for possible changes in binding to the B / E receptor of HeLa cells. Fig, 5 shows the binding of Lp-B as compared to LDL isolated by ultracentrifugation (1.030 kg/l < d < 1.053 kg/l). The molar ratio of apolipoprotein E to apolipoprotein B , was determined by enzyme immunoassay to be 0.25 in the LDL fraction used as a reference. The apoC-11I content was close to zero. LDL and Lp-B were compared at the same apoB content, assuming one apoB per particle. In other words, results express the number of bound apoB containing particles as a function of the number of particles present in the medium. Apparent K a values were 18,9 nmol/I for Lp-B and 10.5 nmol/I for LDL.

% 5O "1 40 t

Large-Lp B

30

lO o

< 1 0 1 0 14 18 2 2 26 30 34 88 42 46 50 5 4 > 5 4

Discussion This work represents the firsl detailed characterization of Lp-B obtained from whole plasma of normolipidemic humans. This study demonstrates that the use . f sequential immunoaffinity chromatography with anti apoB, anti apoE, anti apoClll and anti apo(a) on normolipidemic plasma allows the preparation of lipoproteins containing a large majority of particles virtually free of non apoB protein. However, it is interesting to note that in most cases, some particles containing apoB associated with apoA-I and/or apoA-ll and/or apoA-IV were still present after removal of apoC-lll, E and (a). Improvement of this immunoaffinity method to get large quantities of these distinct particles should help to characterize them and to determine their clinical importance, In contrast with the method recently pub-

nm

rim

% 50 4o

" a-

2O 10 0 54 nm

Fig, 5. SizedistObutMn of whole Lp-B, Large Lp-B(¢lulion volume 8-tl.5 ml),$nzatlLp-B(elution volume 11.5-15.5ml)calcuMtcdfrom electron micrc~copy. Re~uhsare e x p r e s s e d as the pcrcentag~ of the total number of measured particles{500).

175 Bound;Free

1

fmeles aim B/ mg cell proteins 100 '

Im ]-pB |

i-~!

80' 60 4O 20 0 10

i 20

~t

i 30

40 nmoles apo B/I

Fig. 6. (a) Specificbinding of I~I-LDL and Iz"I-Lp-Bto HeLa ceils, difference between ~olalbinding and unspccihc binding was obtainnd with a 20-fold exce~ of unlabe[Icd LDL. Resutt~expt~,s the number of bound apoB cont~ning particles~ a ruction of the number of particl~ p r i n t in the medium, a~umlng one mol~ule of apoB per particle. (b) Scalchard plot repr~3entationof the binding data. Bound/F~: the amounl ot bound 125[-LDL~r I2~I-Lp-Bdivided by the amount of unbound lipop~tcin in the r~clion mixture. concentrations reflects the absence of dissociation during the purification step. Although most of the plasma apoB-containing lipoproteins with densities less than 1,019 kg/I are associated with apoE a n d / o r apoC peptides [21-281, Lp-B particles were also detected in the VLDL regi~.n [29]. Some of these particles have also been identified in the H D L density range [30-31]. Nevertheless Lp-B particles were described mainl) in the density range 1.019-1.063 kg/I [251. The size frsetionation of Lp-B prepared by sequential immunoaffinity chromatography confirms these results and demonstrates that most of the Lp-B particles are present in the LDL size range, while a few particles are largec and a few are smaller. This size heterogeneity of Lp-B particles r0ases the question of their origin. It has recently been shown that HepG 2 cells can secrete lipoproteins containing only apoB (Lp-B) and others which contain both apoB and

apoE (Lp-B:E) [32]. These Lp-B particles are triacylglycerol rich with cholesterol ester representing about half of their total cholesterol mass. They were identified in VLDL, LDL and HDL and their size ranged from 10 to 35 nm. It has been suggested [321 that in plasma, these secretory products may interact with apoC peptides derived from apoA-I and apoA-ll containing lipoproteins of high densities, to form Lp-B:C and Lp-B : C : E particles. These resulting products may undergo hydrolysis eataly'zed by lipoprotein lipase. This lipolytie degradation may lead to the formation of cholesterol ester rich Lp-B particles as the main product [24, 33, 34]. The size heterogeneity of Lp-B particles isolated from whole normolipidemic human plasma may reflect this metabolic process. Results indicate that large Lp-B particles are richer in triacylglycerol and poorer in cholesterol esters than small Lp-B particles. It is probable that this difference in lipid composition reflects a

176 difference in the metabolic stage of these particles. Large Lp-B particles m a y presumably be liver secretory products while small Lp-B particles could be mostly the result of lipolytic d e g r a d a t i o n of m o r e c o m p l e x lipoprotein particles a n d / o r larger Lp-B particles, T h e continuous size spectrum could result from the presence in t h e preparation of particles caught at different stages of their metabolism. T h e observed differences in the lipid c o m p o s i t i o n between isolated L p - B particles m a y d e p e n d o n the metabolic state of the isolated particles, I n o t h e r words, high T G / T C ratios m a y reflect the presence of significant a m o u n t s of large L p - B particles. T h e remarkable stability of the P / T C ratio m a y be related to a lower sensitivity of phospho!ipids to lipolysis. L p - B purified a c c o r d i n g to the procedure described in this p a p e r exhibited a lower affinity to the L D L (apoB, E) receptor than L D L (1.030 kg/1 < d < 1.053 k g / 1 ) . T h e difference m a y be d u e 1o the presence o f a p o E in the L D L isolated by ultracentcifugadon. T h e discrepancy between o u r results a n d those published elsewhere [4] m a y be due to difference in the ways in which the results are expressed a n d / o r differences in the c o m p o s i t i o n of L D L used as a reference. It should be e m p h a s i z e d t h a t o u r results express the n u m b e r of b o u n d particles as a function of the n u m b e r of particles present in t h e m e d i u m , assuming o n e molecule of a p o B p e r particle. T o express results as a function of total protein c o n t e n t m a y be misleading in e x p e r i m e n t s c o m p a r i n g b i n d i n g properties of lipoproteins w i t h different apolipoprotein compositions. Moreover, it should b e kept in m i n d that o u r results were o b t a i n e d w i t h w h o l e Lp-B, in contrast to the p a p e r published before [4]. Variations in the ratio between large a n d small L p - B couM lead to different b i n d i n g capacity. I t w o u l d be o f gieat interest to s t u d y the b i n d i n g properties of L p - B particles in the different size ranges. Nevertheless the conservation of the b i n d i n g properties suggests t h a t affinity c h r o m a t o g r a p h y preserved the integrity a n d the biological properties of the isolated particles. In s u m m a r y , this p a p e r demonstrales that it is possible to isolate Lp-B particles from whole n o r m o l i p i d e m i c plasma, using sequential i m m u n o a f f i n i t y c h r o m a t o g r a p h y w i t h anti apoB, a p o E , a p o C - I l l a n d ape(a), eventually completed by a further purification over a n a n t i a p o A - l , a p o A - l l o r a p o A - l V column. These particles exhibit a size heterogeneity w h i c h m a y correspond to different origins or metabolic states of these particles. Acknowledgments We acknowledge Dr. S h e p h e r d for helpful assistance in manuscript preparation a n d Mr. D e r u d a s for his skillful technical assistance.

References I Alaupovlc. P. (10821 La Rieerca. Clin. Lab. 12, 3-21. 2 MeConathy, WJ., Koren. E., Wieland, H., Camp~, E.M., Lee, D.M., Kloer, H.U. and Alaupovic, P. (17~;5)J. Chromalogr. 342. 47-66. 3 Castro. G.R. and Fielding. C.J. (1984) J. Lipid. Res. 25. 58-67. 4 Zcchner, R., Moser, R. and Kosmer, G.M. (1986) J. Lipid. Res. 27. 681-686 5 Vu-Dae. N.. Mezdour. H , Parra~ H, Luc, G.. Luyeye. L and Fruchan, LC. (1989) J. Lipid Res., 30.1437 1444. 6 Mezdour. H., Clayey, v . Kora, I.. Koffigan, M., Barkia. A. and Fruehart, J., C. (1987) J. Chromatogr. 414, 35 45. 7 Vaitukailis, J.. Robbins, J.B., Nie~ehlag. E. and Ross, G.T. (197l) I. Clin. Endc~rinol. 33, 988-991. 8 Steinmet~ A., Clayey, V., Vu-Dac, N., Katfaruiek, H. and Fmchart. J.C. (1989) J Chromatogt. 487. 35-40. 9 Fruehart, J.C., Kor& I., Cachera, C., Clayey. V., Duthieeul, P. and Mo~chetto, Y, (1982) Clui. Chem. 28, 59-62. 10 Fmch~rt, LC. Filet, C. and Puchols. P. (1985) in Methods of enzymatic analysis (Bergmeyer, H.U., ¢d.) (3rd edn I. pp. 126-138, V~lagsgescllschaft, Weinheim, 11 Lowry, O.H., Roscbrough, N,J.. F~r, A.L and Randall, R.J. (1951), J. Biol. Chem. 193, 265-275. 12 Weber. K. and Osbora. M (I969) J. Biol. Chem. 244, 4406-441-/. 13 Towhin. H.~ Staebelui, T. aria Gordon, J. (195'9) Prec.. Natl. A~d. St:i. USA 76, 4350-4354. 14 Havel, R.J., Eder, H.A. and Bragdon, J,H. (1985) J. Clin. Invest, 34,1345-1353. I5 McFarlane, A.S. (1958) Nature. 182, 53-57. 16 Bilh~mar, D.W., Eisenbcrg. S, Lacy, R.I. (1972) Bi~hem. BiD phys. Acta. 260, 212-217. 17 Johnston, D., Robson, J.M. and Me!nykovich, G. (19831. Endo¢rlnology 113, 90/-914. 18 Goldsiein, J.L., Basu, S.K., Brunschtale, G.Y. and Bi'lr~'n, M.S. (1976) CuB. 7, 85-95. 19 Kane, J.P, Hardman, D,A, and Paulus, H.E. (1981) Pr~. Natl. Acad. Set. U.S.A. '77, 2365-2369. 20 S¢.atchard, O. (1949) Ann. N.Y. Aead. S¢i. 51, 660-672. 21 Osborne, J.C., Jr. and Brewer, H., B., Jr. (1977) Adv. Ptoleui. Chem. 31,253-337. 22 Alaupovic, P, (1972) Protides. Biol. Fluids. 19, 9-19. 23 Alaupovie, P., Lee, D.M. and MgConathy, WJ. (1972) Bioghim. Bio~hys. Acta 260, 6g9-707. 24 Alaupovlc, P., Wang, C,S., McConathy, W..I., W e i r , D. and Downs, D. (1986) Arch. Bi~hlm. Biophys. 244, 226-237. 25 L¢¢, D.M. and Alaupovic, P. (19741 Buichem. g. 137, 155-167. 26 Curry, M.D., McConnathy, WJ. and Alavcovic, P. (19"/7) Biochlm. Biophys, Acla 439. 413-425. 27 Fellin, R , Agosani, B., Rust, W. and SeideL D. (1974) Clin, Claim, Ae 54. 325-333. 28 Patsgh. J.R,, Sailer, S. and Braunsieiner, H. (1975) Eur. J, Clin. invest. 5, 45-55. 29 Alaupovic, P. (1984) presented at Ihe IVth PanAmedcan Congress of Biochemistry. Buenos Aires. Argemma, November 4 - ~ 1984; Abstract 145. 30 Ko~mer, G. and Alaupovic, P. (1932) Biochemistry. 11, ~19-3428. 31 Kqsmer, G. (1972) Biochem. J. 130. 913 917. 32 Da~hti, N. Alaupovi¢, P, Kmght-Gibson. C. and Koren, E. (1987) Biochemistry. 26, 4837-4846. 33 Eisenherg, S., Chajek. T, and Deckelhaum, R. (1978) Pharmaeol. Res. Commun. t0, 729-738. 34 Tam, S,P. and Breegenrldge, W.C, (1983) J, Lipid. Res. 24,13431357.