Two-dimensional electrophoresis of plasma lipoproteins: Recognition of new apo A-I-containing subpopulations

Biochimica et Eiophysica Acta, 1169 (1993) 291-300 0 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2760/93/$06.00

291

BBALIP 54246

Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apo A-I-containing subpopulations Bela F. Asztalos, Charles H. Sloop, Laurence Wong and Paul S. Roheim Diuision of Lipoprotein Metabolism, Department of Physiology Louisiana State University Medical Center, New Orleans, LA (USA)

(Received 13 May 1993)

Key words: Reverse cholesterol transport; HDL, Coronary heart disease; Prep apo A-I; Subpopulation: Plasma lipoprotein;

Electrophoresis, two-dimensional; Ultracentrifugation Two-dimensional electrophoresis has been used to resolve 12 distinct apo A-I-containing high-density lipoprotein (HDL) subpopulations in human plasma. The subpopulations were quantitated by 1251-labeled, monospecific antibody and phosphorimaging. Modification and standardization of the agarose electrophoresis (first dimension) enabled us to recognize new HDL subpopulations. Lipoprotein mobilities in agarose were expressed relative to the mobility of the sample’s endogenous albumin. We demonstrated the presence of lipoproteins with mobilities faster than and similar to albumin, as well as subpopulations with mobilities slower than albumin. We refer to these as precu, a! and prep, respectively. Lipoprotein molecular sizes were determined with a non-denaturing polyacrylamide gradient gel electrophoresis (PAGE) (2% to 36%) in the second dimension. Internal standard of 1251-labeled proteins of known molecular size was run simultaneously in each gel permitting accurate size determination. We have demonstrated that ultracentrifugally-isolated lipoproteins are different from the native apo A-I-containing subpopulations. The major difference observed was the loss of prep, and prep, particles from the d < 1.21 g/ml fractions to the d > 1.21 g/ml fractions. Possible physiologic and pathologic implications of these findings are also discussed.

Introduction Numerous studies have shown a negative correlation between plasma high-density lipoprotein (HDL) concentrations and the risk of coronary heart disease (CHD) [l-5]. This relationship is thought to be due to HDL’s role in reverse cholesterol transport [6-S]. HDLs are not homogeneous and can be separated into several subpopulations [9-111. A correlation between HDL subpopulations and CHD [12-131 has also been documented. Fielding and co-workers demonstrated the heterogeneity of HDL and provided evidence for the physiologic role of HDL subpopulations in reverse cholesterol transport by employing two-dimensional electrophoresis [8,14-161. This paper describes a standardized system of twodimensional electrophoresis that enables quantitative evaluation of apo A-I-containing HDL subpopulations. These studies provide evidence for the existence of apo A-I-containing HDL subpopulations not previously de-

Correspondence to: P.S. Roheim, Division of Lipoprotein Metabolism and Pathophysiology, Department of Physiology, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, LA 70112, USA.

scribed, which may have physiologic roles in lipid homeostasis and reverse cholesterol transport. Materials and Methods Processing of blood

Fasting blood was collected from four male and four female normolipidemic human subjects and stored on ice with the following additives: ethylenediaminetetraacetate (EDTA), 1.2 g/l, sodium azide, 0.1 g/l, gentamicin sulfate, 80 mg/l and Kalikrenin inactivator (aprotinin), 10.0 KU/l as a final concentration. After the plasma was separated from the blood by centrifugation, phenylmethylsulfonylfluoride (PMSF) and benzamidine, (1 mM each, final concentrations) in isopropanol were added l-171.Plasma was stored on ice for short-term use (l-2 days) or frozen at -70°C. Frozen plasma was thawed at 37°C and kept on ice for further analyses. For selected studies, plasma lipoproteins were isolated by ultracentrifugation using a Beckman ultracentrifuge (Fullerton, CA) with an SW60 rotor and spun for 48 h at 40000 rpm 1181.The density of the plasma (2 ml) was adjusted to 1.25 g/ml and then overlayed with a d = 1.21 g/ml KBr solution. Following centrifugation,
292

Stc?ck so~atio~s,

(A> Tris, IO.75 g; boric acid, 5.04 g; disookim EDTA, in distilled water to 1006 mi firma1 mide 2.3 g (dissolved in 100 n-n of solution

Agarose electrophoresti (first Dimensions

Tris-tricine buffer (25 mM, pit-6 S.S>, eo~ta~~~~g calcium lactate and 0.05% sodium azi used for both gel and osity agarose, 0.7% 6 o&ford, ME), was cast and electrophoresed u cals GE 2/4 recirculating appar den). To avoid free electrophoresi and the wall, sample channels were for &ally thinned, ten-channel comb in the prevented sample contact with the glass plates. I or 2 to 4 ~1 with Tris-tricine jv~Hof plasma were dil eroI and bromphenol blue buffer containing 10% and were applied to the sample wells. Samples were electrophoresed at constant voltage (250 V) and maind at 10°C with a recirculating cool genous serum albumin stained witk brom lue, ran 3.5 cm. Agarose was removed fr settes and individual strips were cut out.

monk-n persulfate, 0.450 g (disso ml of solution A, freshiy prepared ea s. of the solutions hste 2% gel: 36% gel:

) 2.8 ml (A) ------

(B) 25 mi

(C) 26 ml

03) 20 ml.

(C) 5 nl

(D) 10 ni.

channels. The aga

e

stripwas placed

epm were applied to the internal stan -denaturing po~yac~~~mide gradient gel elecoresis (second dimension) preparation of 2% to 36% gel. A 2% to 36% noncle-

naturing, concave gradient polyacrylamide gel was use to separate apo A-I-containing lipopro ue used was similar to that descr [20] witb the modifications noted below. Con were prepared using a modified gradient former d 395, Rio-Rad, Richmond, CA) where one chamb vertieally partitioned, resulting in a 2 : 1 Cvv/v) ratio for 2% and 36% polyacrylamide. The gels were cast a modified Pharmacia (XC-8 gel slab casting apparatus. To create a uniform gradient, the shape of e bottom of the casting apparatus was modified from flat to a “V” shape. The cassettes were elevated to raise their lower ends to 1.5 cm from the bottom of the casting apparatus. A casting flow rate of 3 ml/min was aintained with a peristaltic pump prairie Hnstru

ed us& a buffer eon orac acid, and 2.5 mM

the transfer

buffer.

o Layers of fiber

Lip

aper. Simiiar to o~ur

293 previous observations [23], we found that membranes from different manufacturers, as well as different lots from the same manufacturer, occasionally resulted in inadequate capture efficiencies. Therefore, capture characteristics for each new batch of membranes were tested. Fixing, blocking, and immunolocalization. To fix the proteins to the nitrocellulose, the membranes were completely wetted with phosphate-buffered saline (0.01 M Na phosphate, 0.145 M NaCl, pH 7-PBS), then incubated in PBS containing 0.03% glutaraldehyde in a plastic container, and rotated horizontally for 10 min at room temperature. Following fixation, membranes were rinsed twice with PBS to remove residual glutaraldehyde. Free protein-binding capacity of the membrane was blocked by a lo-min incubation in PBS containing 0.05% Tween 20 (PBST) with 5% nonfat dry milk with continuous shaking. Apoproteins were immunolocalized by 3 h of incubation with a monospecific goat anti-human apo A-I antisera. Membranes were transferred into a container with goat anti-human apo A-I antisera in 5% milk in PBST, while shaking continuously. After incubation with the primary antibody, membranes were washed three times for 3 min in PBST. Membranes were blocked as described above for 10 min, and subsequently incubated with a secondary affinity-purified iodinated rabbit-anti-goat antibody (Zymed Laboratories, South San Francisco, CA). The specific activity of this antibody was (0.5 to 1.0. lo9 cpm/mg). Approx. 1 pg antibody diluted in 80 ml PBST milk was used. Membranes were incubated for 3 h, followed by four washes of 3 min each, in PBST. Iodination of the secondary antibody was accomplished by the iodine monochloride method [24]. Following quantitation for apo A-I, membranes were re-incubated with anti-human albumin anti-sera prepared in goats in our laboratory (1: 100 000 dilution). This was followed by washing, blocking, and applying radiolabeled anti-goat y-globulin as described above. Membranes were re-run on the PhosphorImagerTM to obtain a reference point for R, determination (Fig. la). Measurements of the distribution of apo A-I-containing subpopulations. Membranes were exposed overnight

to a PhosphorImagerm screen (Molecular Dynamics, Sunnyvale, CA), and the cassettes were imaged by PhosphorImagerTM. Data were expressed as pixel points determined by the computer and were linearly correlated with the dpm of the lzsI bound to the antigen-antibody complex [25]. For size determination, vertical rectangles were constructed through the gradient gel around both the internal standards and particles with (Yand precu mobilities. The star in the middle rectangle indicates the position of human albumin (Fig. la). Radioactivity was integrated by the computer using the Molecular Dy-

namics ImageQuantTM program [251 (Fig. lb). For each subclass, modal diameters were derived using integral curves (Fig. lb). Size was calculated using the internal standards. This was followed by localization of albumin after detection with anti-human albumin, as described above. Using these two coordinates - R, (relative to albumin) and size - each area was delineated and the pixel volume determined. Percent distribution was calculated for each gel. A minimum of four separate gels was evaluated for each sample. Results Detailed description and evaluation of the quantitative 2-D system Agarose electrophoresis (first dimension). Samples run

on a flat-bed agarose electrophoresis system are subject to syneresis r. This can be prevented by efficient uniform cooling; therefore, the agarose was cast into a glass cassette and run in a vertical electrophoresis unit providing better cooling efficiency. To optimize separations and resolutions of apo A-I-containing particles, agarose with three graded electroendosmosities LE (low), ME (medium), HEEO (highest), and HGT (agarose with high gelling temperature) at three different concentrations (0.5, 0.7, 1.0%) were compared using two buffer systems (Tris-tricine, barbital). Optimal resolution for CY,prea, and prep particles was obtained using LE agarose at 0.7% concentration. No differences were found in resolution between the Tristricine and barbital buffers; therefore, the more stable Tris-tricine buffer was selected. The effect of bovine serum albumin (BSA) (0.1% to 0.3% in the agarose) on lipoprotein mobility was tested. Presence of albumin in the agarose changed the electrophoretic mobility of the lipoprotein and interfered with the appearance of the precv particles (Fig. 2); therefore, albumin was not added to the agarose. Fig. 3 compares the electrophoretic mobilities of purified human apo A-I with apo A-I-containing subpopulations of plasma. Purified apo A-I migrated with prep mobility. Non-denaturing polyacrylamide gel electrophoresis (second dimension) and transfer system. End-point of

lipoprotein migration was determined by electrophoresing the samples for 16, 24, 28, and 32 h; after 24 h, proteins with sizes larger than 4.66 nm did not migrate further through the gel. Therefore in these studies, electrophoresis was carried out for 24 h. Accu-

’ Syneresis: agarose releases water to the surface, where the resistance is higher causing local increase in the agarose concentration. This change in agarose concentration interferes with the accurate separation of lipoproteins.

294

Fig. 1. (a: Appearance of apo A-I subpopulations in the second dimension after two-dimensional electropboresis. Piasma was electrophoresed in the first dimension in 0.7% agarose followed by application of the agarose strip to the top of a ~on~c~atur~~g 2% to 36% concave polyacryiamide gel and subsequently electrophoresed. On the left, internal standards are shown. Rectangles were formed around the internal standards and particles with (Yand precr mobilities. The star in the rectangle of a-migrating particles indicates the position of human albumin obtained after immunoiocalization subsequenr to apap A-I ~mmun~loca~~zat~o~. drodynamic diameters of internal standards: !I) ~~yr~~~ob~l~~(17 nm); (2) ferritin (12.2 nm); (3) catalase (9.51 nm); (4) lactate ~~b~dro~~~a§e (8.16 nm~; (5) albumin (7.1 nm); and (6) ova%bumin (4.66 nm). (b) Radioactivity was integrated over the rectangular areas shown in Fig. la. Median size of lipoprotein particles was estimated by comparing their relative bp, to those of the internal standards.

racy of the size determination was a~c~rn~~~s~e using rzI-labeled internal protein standa to 17.0 nm) run at 60000 cpm/sample ch To pour reproducible gradient gels, tb atus was modified as described in the thods section, and a slow 3 ml/mm used. This slow speed decreased t resulted in a more unifo t ~~~~ea~e~the time required for pouri 40 min. For a good quality gel, it was tion times similar and to complete the process in 1 h for both the 2% and 36% acrylamide g&s. achieved by using a TEMED gradient as de the Materials and Methods section The gels were the day before use and stored at 4°C in gel in plastic containers. While the agarose were running, the polyacrylamide gels were warme room temperature.

transfer,

t

embranes were fully dried and stored

the buffer before total imm

.

binding was evaluat

embranes were primary and seconda

re incubatisn withL a~tibod~~$. Three 0% fetal calf serum with ry milk) were ~o~~are~~ the

milk in PBST at room t~~~~rat~~~~ Using 5% nonfat dry milk, adequate blocking of all the excess proteinbinding capaci embrane took 10 min. Incubation time with the ‘mary and Seco ies was also tested fr a to 8 h.

295

Fig. 2. Separation of apo A-I particles on agarose in the presence or absence of albumin. The upper part of the figure is the radioactive image of apo A-I. Lower curves are scans of the radioactive images. (1) prep particles. (2) (Y particles. (3) prea particles.

binding increased with incubation time using either antibody, the primary or secondary. To complete postmembrane manipulations in 1 day, 3-h incubation times for either antibody were selected. To remove unbound proteins after incubation with primary and secondary antibodies, membranes were washed for 3 min, three times and four times, respectively. No additional decrease in the background occurred after this washing procedure. To quantify ‘251-labeled antibody-binding, we used a PhosphorImagerm (Molecular Dynamics). The advantage of this computerized system is its wide range of linearity and greater sensitivity as compared to conventional X-ray films [25]. In addition, data can be stored in the computer for future analysis. The entire procedure for handling the membranes after the transfer is summarized in Fig. 4. Characterization and quantitation of apo A-I subpopulations

Wet the (in

r11

I31

1

(0.03%

f51

k-l

Fig. 3. Comparison of agarose electrophoresis of purified human apo A-I and plasma. Apo A-I was immunologcalized with anti-human apo A-I antisera as described in the Materials and Methods section.

10 min)

in PBST for

10 min)

i

(2 x ;” pBs) A

I71

181

Block

(5% dry milk Incubate

with

1

the first antibody (goat anti-human apoA-I antisera in 5% milk in PBST for 3 hr) I

(3x3

Wash rain with

PBST)

I

Block (5% dry milk in PBST for 10 min) I Incubate with the secondary antibody (rabbit anti-goat IgG sera in 5% milk in PBST for 3 hr) 1 Wash (4x3 min with PBST)

f91

purified

for

Rinse

I61

A two-dimensional coordinate system was developed to identify apo A-I-containing subclasses. In the first

plasma

Fix

glutaralde,hyde

I31

I41

membrane PBS)

1

IlO1

Dry

Ill1

Analysis by PhosphorImager

1131

1

Incubate with (5% milk in Repeat

steps

I

anti-dog PBST for I 6 through

albumin 3 hr) 11 above

Fig. 4. Summary of steps for membrane treatments after transfer.

296 Table 1 Two-dimensional

codinates

nnd percent diseribufbn

First dimension

Second dimemion

(median

Modal diameter

R,)

(2rn!

oj .!,P

.! -I

% Distributio-

ated particles according to their

Twelve separate subpopuBati based on the parameters obtained in the first and dimensions. Fig. 5a shows apo A nsion (agarose electrophe d~st~ib~t~Q~ in the secon below. Fig. 5b provides a SC atic ~e~~ese~tat~~~ of the individual apo A-H s&p ations based on inte~ lb. Table I summagration of areas as shown 1 rizes median R,s for the s~~~Q~~~ati~ns i dimension, modal diameters in the seco as well as percent distribartion of the in s as shown in Fig two particles with small, but distinct, size differences b, 5.38 nm). Three pre odal diameters were prep,,, 12.85 m-n; and pre particles appeared with CI

Values represent the mean + SD. times. SD. = standard deviation.

of eight subjects

deter&n&

f3ur

re routineHy recsgnized: reMJs 8.40 nm; and

eta

I&:

bet

Fig. 5. Two-dimensional separation of apo A-I subpopulations. (a) Plasma was electrophoresed in the first dimension (C.7% agarose) followed by application of the agarose strip to the top of the nondenaturing concave polyacrylamide gel (2% to 36%), and subsequently electrophoresed. This shows apo A-I distribution in the second dimension. The star indicates the position of human albumin obtained after imm~nolocaIiza~~o~ subsequent to apo A-I immunolocalization. The horizontal insert on top represents apo A-I distribution on a duplicate agarose strip. jb) Schematic representation of individual apo A-I subpopulations based on integration of areas as shown i3 Fig. Ib.

297

recombined

d Fig. 6. Influence of ultracentrifugation on the two-dimensional separation of plasma and corresponding d < 1.21 g/ml and d > 1.21 g/ml fractions. Internal standards are on the left in each gel. Stars indicate the location of albumin in the plasma, d > 1.21 g/ml, and in the combined d < 1.21 g/ml and d > 1.21 g/ml fractions. Distribution of apo A-I subpopulations on two-dimensional electrophoresis. Equal amounts of plasma equivalents were applied to each separation. (a) Plasma; (b) d < 1.21 g/ml; (c) d > 1.21 g/ml; (d) d < 1.21 g/ml combined with d > 1.21 &/ml.

precu4, 7.67 nm. Most of the apo A-I was found in the CYand precu particles (Table I>. Reproducibility. To test reproducibility of the method, the same human plasma was applied to three different batches of gel produced at different times. Each batch was electrophoresed and transferred to a nitrocellulose membrane. All membranes were treated under identical conditions. Each membrane was quantified, as described above, and percent distribution was calculated (Table II).

Effect of ultracentrifugal separation on apo A-I subpopulations Two-dimensional separation of the d < 1.21 g/ml and d > 1.21 g/ml fractions was compared to plasma.

A representative

experiment is shown in Fig. 6. In the lipoproteins, only particles with CYand precu mobilities were found. In the d > 1.21 g/ml

d < 1.21 g/ml

Table III (A) Cholesterol and apo A-I distribution between plasma and ultracentrifugally separated fractions

Table II Plasma

Analysis of error for apo A-I distribution Lipoprotein region

Mean %

Intra-assay CV %

Inter-assay cv %

18.7 30.2 17.8 6.3 7.5 3.6 2.5 3.6 4.0 1.2 2.1 1.3

4.9 2.4 11.6 13.0 6.4 20.0 17.8 18.2 13.1 18.1 10.0 13.3

10.6 5.9 14.0 9.5 8.0 19.4 24.0 17.7 14.3 16.6 9.5 23.0

CV = coefficient of variation. Human plasma was applied to three different batches of gel produced at different times. Each batch contained four gels. All membranes were treated under identical conditions.

Cholesterol Apo A-I

167 121

d < 1.21

d > 1.21

mg/dl

mg/dl

141.4 (85) * 86.5 (71) *

Total recovered

3.2

144.6

(2) *

(87) *

22.9 (19) *

109.4 (90) *

IZ= 4. * = percent recovered. (B) Ago A-I distribution between plasma and ultracentrifugally separated apo A-I-containing subpopulations

Title

Plasma

ApoA-I distribution d < 1.21

d > 1.21

mg/dl

mg/dl 11.2+ 1.9

(Y

76.2 + 7.1

63.7+7.1

precu prc& prep,

26.9 + 2.2 8.0 f 1.0 5.7 * 0.4

22.8 f 1.6 0.0 0.0

All values are mean + S.D. n = 4. S.D. = standard deviation.

4.6 f 1.1 6.2 f 0.7 0.9 + 0.3

298 ha&m, particles with pre/3r, prep,, M and prea mobihties were detecte < 1.21 g/ml iipoher partiproteins were different from plas the cles were absent in the ultracent arated agaily and precu-migrating lipoprotein fractions. Plasma sizes were between 13.69 nrn g/n11 sizes were between when. the d < 1.21 g/ml recombine (Fig. e appearance of the opulations as di from n~fra6ti~~ated asma. It is important to note that larger particles were detected in the a’ > 1.21 g/ml fraction &Table HO. To evaluate the influence of ~~trace~tr~f~gat~o~ on ulations, we exmass (Table III). cause the sizes of

ardized to obtain improved resoliution of parricls .n ation enarsled iis to

ty faster than atibumin (Rf > :.; e~~-mig~at~~~parti&s. We inc~a~act~~~~a~~o~of snbpopuiata by relating particie m&i&y (second dimension) cing internal standards of proused a 2% to 36% gel gradient to improve the size resolution. To ehrninate any possibIe mfkuence of

e length of electrophohesis on migration characteristics tions (Table III).

of the major subpopu%a-

Using two-dimensional electrophoresis based on electrophoretic mobility and size, Fielding and associates established the presence of several distinct apo A-P-containing subpopulations [8,11,14-141. Specific physiologic functions of these subpopulations also have been demonstrated [14-J_6]. Several other groups have shown similar, if not identical, however, their methods, and t nomenclature they used to define these subpopulations varies [L&5,27]. have adopted the nomenclature developed by Fielding and co-workers for designating MDL sub~o~~latio~s because their work has provided the most extensive chemical and functional characterizations [l4-161. In addition, we used I, 2, 3, and 4 designations to classify articles based on size for the CY-an s. e made several improvements in the generally method of agarose electrophoresis of li y keeping the condition of the preparation constant, i.e., eliminating syneresis. Briginally, albumin was added to the agarose to “correct unevenraess of the ds” 1281, however, in our system, we found ition of albumin decreased the separation of particles. Et also has been shown that “‘the electrophoretic mobility of a-lipoproteins and albumin are virtually identical” [28]. Therefore, we used the location of endogenous albumin to define mobihties of L particles in the first dimension. To faciiitate further chemical characterization and future physiologic and pathologic studies, we developed a system that better defines WDL subpopu~a~io~s. The procedure for agarose electrophoresis was stand-

not migrate further,

tion of syneresis.

Only occasionahy of the presence of s

e we observed any i opulations with prq3 an be ~t~~~b~te~ to the amount of pIasrna applied; we used 5 to 20 5mes less

299 plasma in our agarose system than other investigators [14]. Because these particles are present in very low concentrations, we may have been unable to detect them; therefore, we have not included these particles in our descriptions of HDL subpopulations. It was important to compare distribution of plasma apo A-I-containing subpopulations determined using our method with subfractions separated by ultracentrifugation. Plasma and ultracentrifugally isolated lipoproteins are different in size and distribution of apo A-I subpopulations (Fig. 5, Table III). Alteration of lipoproteins by ultracentrifugation has been reported previously by several investigators showing that apo A-I is lost during ultracentrifugation [31-351. It is important to be aware that this is a specific loss (Fig. 5, Table III), and it is not a random shearing off of apo A-I. Unique subpopulations are lost during ultracentrifugation from the d < 1.21 g/ml lipoprotein fractions, and then recovered in the d > 1.21 g/ml bottom. We have demonstrated similar losses when centrifugation has been carried out at d = 1.24 g/ml. Thus, the physiologic behavior of the d < 1.21 g/ml lipoproteins may not be identical to lipoproteins in the circulation. It should be emphasized that subpopulations with established physiologic roles (i.e., prep, and prep,) [14161 are not present in the d < 1.21 g/ml lipoproteins. One of the largest apo A-I-containing particles, the prep, subpopulations with modal diameters of 12 nm to 14 nm, did not float at a density of 1.21 g/ml. Prep, particles have been found only in the d > 1.21 g/ml fraction. This suggests that prep, particles may have a unique composition and may be similar to those isolated by Kunitake et al. [261. In conclusion, we have described an improved technique for two-dimensional electrophoresis. Using this system, we have shown the presence of at least 12 apo A-I-containing subpopulations in human plasma. We also provided evidence for the existence of precu subpopulations with electrophoretic mobility faster than albumin. We believe that further investigation of apo A-I-containing subpopulations, in both normal and pathologic conditions, will provide an understanding of the overall function and role of HDL subpopulations. This technique could be extended to improve the knowledge about the physiology and pathophysiology of particles containing only apo A-I and those with both apo A-I and apo A-II [36]. It is also obvious that two-dimensional electrophoresis could be applicable for studies of subpopulations containing other apoproteins, which could provide an entirely new area for the study of HDL lipoprotein metabolism. Acknowledgements

This work was supported by a grant from the US National Institutes of Health, National Heart, Lung,

and Blood Institute (HL25596). The authors wish to thank Ms. Gae Garrard and Ms. Susan Allen for editorial assistance and manuscript preparation. The expert technical assistance of Ms. Colleen Tierney is gratefully acknowledged. References 1 Barr, D.P., Russ, E.M. and Eder, H.A. (1951) Am. J. Med. 11, 480-493. 2 Gofman, J.W., Young, W. and Tandy, R. (1966) Circulation 34, 679-697. 3 Gordon, T., Castelli, W.P. and Hjortland, M.C. (1977) Am. J. Med. 62, 707-714. 4 Lechleitner, M., Miesenbock, G. and Patsch, J.R. (1990) Curr. Opinion Lipidol. 1, 330-333. 5 Stampfer, M.J., Sacks, F.M., Salvini, S., Willett, W.C. and Hennekens, C.H. (1991) N. Engl. J. Med. 325, 325-373. 6 Glomset, J.A. (1968) J. Lipid Res. 9, 155-167. Biophys. Acta 326, I Stein, 0. and Stein, Y. (1973) Biochim. 232-244. 8 Fielding, C.J. (1991) Curr. Opinion Lipidol. 2, 376-378. D.W., Nichols, A.V. and Pan, S.S. (1978) Atheroscle9 Anderson, rosis 29, 161-179. 10 Blanche, P.J., Gong, E.L., Forte, T.M. and Nichols, A.V. (1981) Biochim. Biophys. Acta 665, 408-419. 11 lshida, B.Y., Frolich, J. and Fielding, C.J. (1987) J. Lipid Res. 28, 778-786. F. and Saltissi, S. (1981) Brit. Med. J. 12 Miller, N.E., Hammett, 282, 1741-1744. A.G., Meusing, R.A., Schelesselman, 13 Schmidt, S.B., Wasserman, S.E., Larosa, J.C. and Ross, A.M. (198.5) Am. J. Cardiol. 55, 1459-1462. 14 Castro, G.R. and Fielding, C.J. (1988) Biochemistry 27, 25-29. 15 Francone, O.L., Gurakar, A. and Fielding, C. (1989) J. Biol. Chem. 264, 7066-7072. 16 Francone, O.L. and Fielding, C.J. (1990) Eur. Heart J. ll(Suppl E), 218-224. C. and Scanu, A.M. (1986) Methods Enzymol. 128, 17 Edelstein, 151-155. 18 Have& R.J., Eder, H.A. and Bragdon, J.H. (1955) J. Clin. Invest. 34, 1345-1353. B.R., Huang, S. and Wong, 19 Gaver, A., Weedy, N.K., Maldonado, L. (1992) Clin. Chim. Acta 208, 23-37. 20 Lefevre, M., Lefevre, J.C. and Roheim, P.S. (1987) J. Lipid Res. 28, 1495-1507. 21 Pitts, R.F. (1968) in Physiology of the Kidney and Body Fluids, 2nd ed. (Pitts, R.F., ed.), pp. 54-61, Year Book Medical Publishers, Chicago. 22 Hunter, W.M. and Greenwood, F.C. (1962) Nature 194, 495-496. 23 Lee, L.T., Wong, L., Roheim, P.S. and Thompson, J.J. (1989) Anal. Biochem. 180, 358-361. 24 McFarlane, A.S. (1958) Nature 48, 53. 25 Pickett, S. (1992) Molecular Dynamics Technical Notes 55, l-12. 26 Kunitake, ST., La Sala, K.J. and Kane, J.P. (1985) J. Lipid Res. 26, 549-555. 21 Reichl, D., Hathaway, C.B., Sterchi, J.M. and Miller, N.E. (1991) Eur. J. Clin. Invest. 21, 638-643. 28 Hatch, F.T. and Lees, R.S. (1968) in Advances in Lipid Research, Vol. 6 (Paoletti, R. and Kritchevslq, D., eds.), pp. l-68, Academic Press, New York. 29 Nowicka, G., Bruning, T., Bottcher, A., Kahl, G. and Schmitz, G. (1990) J. Lipid Res. 31, 1947-1963. 30 Schmitz, G. and Williamson, E. (1991) Curr. Opinion Lipidol. 2, 177-189.

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