Prog. Lipid Res. Vol. 30, No. 2/3, pp. 105-138, 1991 Printed in Great Britain. All rights reserved

0163-7827/91/$0.00 + 0.50 ~) 1991 Pergamon Press plc

APOLIPOPROTEIN COMPOSITION AS THE BASIS FOR CLASSIFYING PLASMA LIPOPROTEINS. CHARACTERIZATION OF ApoA- AND ApoB-CONTAINING LIPOPROTEIN FAMILIES el/TAR ALAUPOVIC* Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, U.S.A. CONTENTS ABBREVIATIONS

I. INTRODUCTION II. Tim ISOLATIONAND CRARACTERIZATIONOF APOLIPOPROTEINS Ill. APOLIPOPROTEINSAS MARKERSOF LIPOPROTEINPARTICLES IV. CHEMICAL CLASSIFICATION OF PLASMA LIPOPROTEIN$AND THE CONCEPT OF LIPOPROTE1NFAMILIES

105 105 107 112 I14

V. FRACTIONATIONANDCHARACTERIZATIONOF ApoA- AND APOB-CoNTAINING LIPOPROTEIN FAMILIES

A. Introduction B. Fractionation of ApoA-containing lipoprotein families C. Fractionation of ApoB-containing lipoprotein families VI. METABOLICAND FUNCTIONALPROPERTIESOF ApoA- AND ApoB-CONTAINING LIPOPROTEINFAMILIES VII. LIPOPROTEINFAMILIESIN DYSLIPOPROTEINEM1ASAND ATHEROSCLERO~IS

A. Introduction B. Concentration profiles of ApoA-containing lipoprotein families C. Concentration profiles of ApoB-containing lipoprotein families D. Lipoprotein families and coronary artery disease VIII. CONCLUSION ACKNOWLEDGEMENTS

REFFJ~NCF.S

116 116 118 119 122 125 125 126 127 128 131 132 133

ABBREVIATIONS VLDL--very low density lipoproteins LDL--Iow density lipoproteins HDL--high density lipoproteins VHDL--very high density lipoproteins Apo--apolipoprotein LP--lipoprotein LPL--lipoprotein lipase LCAT--lechithin:cholesterol acyltransferase CETP--cholesteryl ester transfer protein

I. I N T R O D U C T I O N

Soluble plasma lipoproteins constitute a unique class of conjugated proteins, the main purpose of which is to transport water-insoluble neutral lipids and phospholipids from their sites of formation to their sites of utilization either as a source of energy or as cellular building components. Machebouef and his coworker were the first to establish the constancy of lipid-protein composition and the presence of specific lipid-binding proteins as the essential chemical criteria for defining individual plasma lipoproteins) ss However, the first classification of plasma lipoproteins was based on their nonspecific physical properties rather than specific lipid-binding protein constituents. The separation of plasma lipids in free electrophoresis into two distinct bands with the mobilities of 0tl- and t-globulins** not only confirmed Nerking's postulate, at the beginning of this century, that *Corresponding address: P. Alaupovic, Ph.D., Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104, U.S.A. I05

106

P. ALAUPOVIC

all plasma lipids are chemically bound to proteins |89 but also led to the notion that they exist as components of two distinct lipid-protein complexes. The concept of two distinct lipoprotein classes was further strengthened by development of a first reproducible preparative procedure for the isolation of human plasma lipoproteins. Fractionation of human plasma in ethanol/water mixtures at low temperature and low ionic strength resulted in the separation of two lipoprotein fractions differing significantly in their solubility properties, size, shape, lipid content and electrophoretic behavior. 67,~°8Because of their characteristic mobilities in the electric field, these two lipoproteins were designated as ~t- and fl-lipoproteins. Thus, the electrophoretic mobility was introduced as the first principal criterion for the operational classification of plasma lipoprotcins which was extended with the advent of zonal electrophoresis to four lipoprotein classes including those remaining at the origin and those migrating to the ~-, r - and pre-flpositions. 71,~49 Due to the presence of neutral lipid and phospholipid constituents, plasma lipoproteins have relatively low hydrated densities in comparison with simple proteins. This characteristic physical property has been successfully used for the preparative isolation of plasma lipoprotcins and as a major operational criterion for their characterization and classification, m Based on Pedersen's application of ultracentrifugal flotation technique to the isolation of a fl-lipoprotein from human plasma, 2m°Gofman and his coworkers demonstrated by fractional ultracentrifugal flotation of lipoproteins in salt solutions of successively increased densities that plasma lipoproteins represent a wide spectrum of particle sizes and densities unequally distributed along a density gradient from 0.92 to 1.25 g/ml. 74'~°3'~°4Based on a profile of minimal and maximal concentrations along this density gradient, lipoproteins were divided into five major density classes designated as chylomicrons (d < 0.94 g/ml), very low density lipoproteins (VLDL, d = 0.94-1.006 g/ml), low density lipoproteins (LDL, d = 1.006-1.063 g/ml), high density lipoproteins (HDL, d = 1.063-1.21 g/ml), and 1.21 infranatant fraction or very high density lipoproteins (VHDL, d > 1.21 g/ml). Studies on the macromolecular distributions showed that each lipoprotein density class represents a polydisperse system of particles heterogeneous with respect to size and hydrated density.81'162'2°2The relatively high degree of this heterogeneity, frequently reflected in the spreading of otherwise symmetrical lipoprotein boundaries in the analytical ultracentrifuge, necessitated further subdivision of VLDL, m'152'2°~'223 LDL 9°'~54'~57'2°4'219'235and HDL 25'H5'2°9'225 into several density subclasses. The utilization of hydrated density as a criterion for the fractionation and identification of plasma lipoproteins has revealed an unsuspected heterogeneity of this system of macromolecular particles. Studies on the macromolecular distributions and lipid and protein composition showed that the polydisperse character of each density class or subclass was due to changing proportions of neutral lipids and phospholipids and changing lipid/protein ratios of individual lipoprotein particles.25'81'90"l11,115,152,154,157,162,193,202,204,208,209,219,223,225,235,243 The results of electrophoretic and immunological studies indicated the occurrence of two distinct proteins; one of these proteins, referred to as a-protein, was found to be characteristic of - or high density lipoproteins and the other, referred to as fl-protein, of fl- or low density lipoproteins. 1'47'1°8'149'|58'226'268Conceptually, plasma lipoproteins were viewed as a macromolecular system of lipid-protein complexes of noncovalently bound neutral lipids, phospholipids and two specific proteins (~- and fl-protein) forming discontinuous particle distributions heterogeneous with respect to hydrated density, size, electric charge and lipid-protein composition) Furthermore, despite recognized compositional and structural heterogeneity, major lipoprotein density classes have been considered and accepted as the fundamental physical-chemical and metabolic entities of the system. Some of the possible reasons for the acceptance of this conceptual view were the emphasis on lipids as the potential injurious agents in the genesis and development of atherosclerosis) °3 the availability of a relatively simple methodology for preparative isolation of lipoprotein density classes 74 and the already developed methodology for quantifying cholesterol,245 triglycerides263and phospholipids. 9~Two additional factors contributing to this conceptual view were the disclosure of a metabolic relationship between major lipoprotein density

ApoA- and ApoB-containing lipoprot¢in families

107

classes 1°2'1°3,j93 and their clinical usefulness for characterizing and classifying hyperlipoproteinemias.~°3'~27'193 Although it had been recognized since the successful preparative isolation o f ~- and fl-lipoproteins by Cohn and his coworkers~7,~°s that each of these two lipoprotein classes contains a distinct protein moiety, the terminal amino acid analyses indicated that, at least, chylomicrons and VLDL may contain additional protein constituents. 2~sm7 Moreover, results of several immunochemical analyses suggested antigenic heterogeneity of all lipoprotein density classes. ~,47,~°~ These studies, in conjunction with a general lack of qualitative and quantitative data on the protein constituents of lipoproteins (apolipoproteins), created in the early sixties considerable interest and need for a better understanding of the role of apolipoproteins in the structure and metabolism of lipoproteins. This newly created interest further enhanced by characterization of inherited hypolipoproteinemias as apolipoprotein deficiency diseases96'222 ushered the lipoprotein field into a new developmental phase that culminated with the discovery of several new apolipoproteins and recognition of their structural and functional properties. II. THE ISOLATION AND CHARACTERIZATION OF APOLIPOPROTEINS The N-terminal amino acid analyses establishing glutamic acid as the only terminal amino acid of LDL and aspartic acid of HDL provided additional evidence that these two major lipoprotein density classes contain single but distinct apolipoproteinsfl a3~ Traces of other N-terminal amino acids in these two lipoprotein density classes were considered as contaminations resulting from incomplete centrifugation of lipoproteins. However, the identification of threonine and serine as the major and aspartic acid and glutamic acid as the minor N-terminal amino acids of chylomicrons and VLDL suggested that these lipoproteins may contain several apolipoproteins. These results offered several possible interpretations as to the number, localization and even authenticity of potentially distinct apolipoproteins. We approached this problem on the assumption that VLDL might consist of several lipoproteins differing in their density properties and specific apolipoprotein composition. 2 However, the N-terminal amino acid analyses of five chylomicron and VLDL subfractions showed that none of these subfractions contained a single protein and that the previously established protein heterogeneity of VLDL applied to each segment of this density range (d < 1.006 g/ml). The failure to identify and separate VLDL subfractions characterized by distinct apolipoprotein composition indicated that further studies on the identification of VLDL apolipoproteins should be performed with deiipidized preparations of this density class. Since the available delipidization procedures applied to VLDL either failed to yield water-soluble proteins 226 or resulted in poor recoveries of lipid-protein residues, 2s'117'227 Gustafson developed a new procedure for partial delipidization of triglyceride-rich lipoproteinsJ °9 In this procedure, lipoproteins were lyophilized in the presence of starch, the dried mixture of lipoproteins and starch was extracted several times by n-heptane at - 12°C and the resulting phospholipid-protein residues were extracted by aqueous buffers. All neutral lipids and some phospholipids were removed. The protein recovery was almost complete for HDL, relatively high for VLDL (40-90%) but very low for LDL (10%). Partial delipidization of VLDL from hypertriglyceridemic subjects yielded a mixture of phospholipid-protein residues which were separated by Pevikon zone electrophoresis and ultracentrifugation into three fractions differing in their sedimentation coefficients, hydrated densities, N-terminal amino acids, peptide patterns and immunologic specificity. ,t0,1,2 Comparison of these characteristics with those of partially delipidized LDL and HDL revealed that the protein moiety of one of the phospholipid-protein residues was chemically and immunologically identical to that of LDL and the other to that of HDL. However, the protein moiety of the third phospholipid-protein residue was characterized by threonine and serine as N-terminal amino acids, and peptide patterns different from those of LDL and HDL proteins. It gave no precipitin reaction with antisera to HDL, LDL, albumin or gamma-globulins, but displayed a single precipitin line with antisera to VLDL or total human serum. I n accordance with Oncley's suggestion that the protein

108

P. ALAUPOWC

moiety of H D L or a-protein be referred to as apolipoprotein A (ApoA) and that of L D L or #0-protein as apolipoprotein B (ApoB), we proposed that the newly recognized V L D L protein be called apolipoprotein C (ApoC). ~I°'H2 The phospholipid/protein ratio of Apo-C-containing residue was approximately three times higher than those of ApoA- or ApoB-containing residues, suggesting a relatively high binding capacity of A p o C for phospholipids. At that time, the proposed models for the structural arrangements of proteins and lipidsin chylomicrons and V L D L considered the central triglyceridemass to be stabilizedby a surface layer of phospholipid and protein with cholesteroland cholesteryl esters as interposed components. 77,2sBecause of its high phospholipid binding capacity, we have suggested that A p o C may play an important role in maintaining the structural stabilityof protein-poor and trigiyceride-richchylomicrons and V L D L . H2 The finding of two terrninal amino acids had indicated either a protein consisting of two nonidentical polypeptides or two separate proteins. Further studies suggested that A p o C might consist of, at least,two separate polypeptides characterized by high mobilities on basic polyacrylamide gel electrophoresis.178 However, Brown et al.~'49 demonstrated that A p o C was composed of three nonidentical polypeptides initiallynamed according to theirC-terminal amino acids apoLP-Val, apoLP-Glu and apoLP-Ala. ApoLP-Val was subsequently corrected to apoLP-Ser Hg'~s3and apoLP-Ala was shown to occur in three isomorphic forms differing with respect to the number of neuraminic acid residues.49 The apoLP-Ser and apoLP-Glu polypeptides were characterized by threonine and apoLP-Ala by serine as their N-terminal amino acids. Thus, despite some initial skepticism regarding the existence of A p o C , 9v'2°3'26mthis apolipoprotein identified as a cluster of three polypeptides was firmly established as an integral protein component of lipid transport system. Despite the early evidence indicating the presence in HDL of a single protein, terminal amino acid analyses,237 immunological teStS, m'32'159and measurement of the protein mass and molecular weights of five HDL subfractions2° suggested a polypeptide heterogeneity of HDL. Subsequently, Shore and Shore demonstrated that the protein moiety of HDL and its HDL2 (d = 1.063-1.125 g/ml) and HDL3 (d = 1.125-1.21 g/ml) subfractions consists of two nonidentical polypeptides called, according to their C-terminal amino acids, apoLP-Thr and apoLP-Gln. 23s'239This important discovery, confirmed by several investigators, 55't4~'22m'22s also provided an explanation for the initial failure to recognize the presence of two major HDL apolipoproteins. The apoLP-Gln was found to have blocked N-terminal amino acid and, thus, could not be identified solely on the basis of N-terminal amino acid analysis. The apoLP-Thr characterized by aspartic acid as its N-terminal amino acid was subsequently found to have giutamine rather than threonine as the C-terminal amino acid and was renamed apoLP-Gln. TM In contrast to studies on VLDL and HDL apolipoproteins, there was no evidence for the polypeptide heterogeneity of the major LDL protein moiety characterized by glutamic acid as its single N-terminal amino acid. The identification of ApoA-polypeptides, ApoC-polypeptides and ApoB as specific, integral constituents of plasma lipoproteins, the occurrence of polymorphic forms of apoLPAla and the possible, if not predictable, future recognition of additional apolipoproteins necessitated the introduction of a nomenclature capable of expressing adequately the relationship between the already identified and newly discovered apolipoproteins, polypeptides and their polymorphic forms. To avoid any ambiguities of a nomenclature based on either terminal amino acids or major amino acids, we have introduced the so-called ABC nomenclature in which apolipoproteins are designated by capital letters, their constitutive polypeptides by Roman numerals and the polymorphic forms of either apolipoproteins or polypeptides by Arabic numbers? '4'7 The main protein of HDL was named ApoA and its nonidentical polypeptides ApoA-I (apoLP-Gln-I) and ApoA-II (apoLP-Gln-II). The main protein of LDL was called ApoB, while the characteristic apolipoprotein of VLDL was referred to as ApoC and its three nonidentical polypeptides as ApoC-I (apoLP-Ser), ApoC-II (apoLP-Glu) and ApoC-III (apoLP-Ala). The three polymorphic forms of ApoC-III were named ApoC-III-0, ApoC-III-1 and ApoC-III-2. We have suggested that any potential apolipoprotein or polypeptide should conform to certain criteria before being recognized as in integral constituent of the lipid transport and incorporated into this

ApoA- and ApoB-containinglipoprotein families

109

system of nomenclature.4'7 An apolipoprotein was defined as a lipid-binding protein (single polypeptide or multiple polypeptides) with the capacity to form a system of soluble, polydisperse lipoprotein particles. The recognition criteira for an apolipoprotein included: (1) unique chemical, physical and immunologic properties, (2) capacity to form lipoprotein particles, and (3) recognition as a distinct structural and/or functional component of the lipid transport system. A criterion specific for a constitutive polypeptide was the capacity to associate with another polypeptide(s) in the formation of an apolipoprotein. Thus, in their lipoprotein form, constitutive polypeptides should give an identity reaction in double diffusion analysis against their corresponding monospecific antisera. Rapid advances in the chemistry of apolipoproteins had a marked impact on the conceptual view of lipoproteins by shifting the emphasis from lipid to apolipoprotein constituents and underlying the significance of apolipoproteins as the most probable determinants of the compositional specificity and structural stability of lipoproteins. One of the immediate consequences of this new trend was the search for and discovery of several additional apolipoproteins. A minor apolipoprotein, first detected by immunological analysis in VHDL ~7and LDL ~s4and referred to as "thin-line" polypeptide, was later shown to occur mainly in HDL. 179This polypeptide called ApoD was shown to be a glycoprotein with a carbohydrate moiety accounting for 18% of its dry weight. ~s~A similar polypeptide was also described by Kostner; ~4°due to its presence in HDL and association with ApoA-I and ApoA-II, this polypeptide was named APOA-III. Despite some discrepancies in the amino acid composition, APOA-III and APOD were considered to be identical apolipoproteins. Albers e t al. 24 confirmed the originally described amino acid composition 179'lsl and this polypeptide remained recognized as ApoD. The isolation from triglyceride-rich lipoproteins of another minor apolipoprotein was reported independently by three groups of investigators. 234'um;59Because of its relatively high arglnine content, this apolipoprotein was initially called the "arglnine-rich" polypeptide. This polypeptide present mainly in triglyceride-rich lipoproteins and HDL is now named ApoE. Olofsson e t al. 2°~ isolated from HDL a minor apolipoprotein characterized by a relatively low isoelectric point (pI = 3.7). This acidic polypeptide present mainly in HDL and VHDL was termed ApoF. Ayrault-Jarrier e t al. 3~ described the isolation from HDL and VHDL of a minor polypeptide that differed on the basis of its immunologic properties, mobility on urea-polyacrylamide gel electrophoresis and amino acid composition from other HDL apolipoproteins. This glucosamine-containing polypeptide was called ApoG. The next two identified apolipoproteins share some similarities regarding their association with lipids and distribution along the lipoprotein density spectrum. One of these two apolipoproteins was first identified in the rat HDL and, because of its presence in this density class, called ApoA-IV. 252 A homologous protein was detected in human mesenteric lymph chylomicrons27° and plasma VLDL of nonfasting hypertriglyceridemic patients. 3s In humans, ApoA-IV was found to be present in its lipoprotein form mainly in lymph chylomicrons and only sparsely in plasma triglyceride-rich lipoproteins (d < 1.006 g/ml) of healthy subjects with mild alimentary chylomicronemia.261 However, more than 90% of ApoA-IV was found to be present in plasma in its lipid-free form. ~°6'26~ The re-examination of ApoA-IV distribution in major lipoprotein classes isolated by 6% agarose gel chromatography rather than sequential ultracentrifugation showed that 15-25% of the total plasma APOA-IV was, in fact, associated with small HDL particles;4~ thus, at least, a portion of lipid-free ApoA-IV was considered to be an artifact of ultracentrifugation. Upon entering systemic circulation, ApoA-IV located on the surface of lymph chylomicrons appears to be dissociated during their lipolytic degradation and, at least, partially transferred to and associated with HDL particles. It has also been suggested that some small HDL particles containing ApoA-IV and ApoA-I as their protein constituents might be of hepatic rather than intestinal origin.4~ The other protein mimicking the distribution of ApoA-IV among major lipoprotein density classes is ~2-glycoprotein I, a plasma ~-globulin first reported in its lipid-free form by Schultze e t al. 232 Burstein and Legmann identified//2-glycoprotein I as the plasma factor necessary for precipitating triglyceride-rich lipoproteins by sodium dodecyl sulfate and suggested that it may play a role in the VLDL metabolism. 5~In their

110

P. A~uPo~c

studies on the polymorphic forms of ApoE, Polz et aL~13 identified fl2-giycoprotein I as a protein constitutent comigrating with ApoE on tetramethylurea polyacrylamide gel electrophoresis and demonstrated its occurrence as an integral protein constituent of VLDL isolated from normolipidemic subjects. Like ApoA-IV, 65-75% of the total fl2-glycoprotein I was found to be present in the lipid-free form with ca. 7-9% occurring in chylomicrons and VLDL, 1-2% in LDL, and 16-18% in HDL. 2H After a fatty meal, the increase in plasma levels of fl2-glycoprotein I (8-9%) was due entirely to its increase in chylomicrons, suggesting the possibility that p2-glycoprotein I might be newly formed and/or secreted during the synthesis of intestinal lipoproteins. 2H Because of its association with lipoproteins, high affinity for triglyceride-rich lipid emulsions212 and its capacity to enhance the ApoC-II activation of lipoprotein lipase, ~Ssfl2-glycoprotein I was considered to satisfy all of the criteria to be classified as an apolipoprotein and was called Aport. Several investigators reported the presence in HDL of protein constituents generated in response to specific stimuli such as the administration of antibiotics in case of the so-called "threonine-poor" polypeptidesTM or parenteral glucose administration in case of "glucoseinduced" or S-peptides. t69'~7°These polypeptides appear to be similar, if not identical, to the serum amyloid AA proteins referred to as SAA proteins m and initially detected as HDL protein constituents in patients affected with severe trauma or a variety of other pathological conditions. 79's°'171'175 Because these polypeptides only occur in negligible concentrations in plasma lipoproteins of normal, asymptomatic subjects, the major question raised by their discovery was whether or not they should be considered as integral components of the lipid transport system. The arguments in favor of considering SAA or S-polypeptides as an integral apolipoprotein include its presence in the plasma lipoproteins, a highly significant increase in various pathological conditions, lipid-binding capacity, occurrence in all major lipoprotein classes, capacity to displace ApoA-I upon incubation with HDL, and the formation of lipoprotein particles in association with ApoA-I. TM A kinetic study with S-polypeptide in healthy subjects indicated the possible existence of two lipoprotein particles with different turnover rates; ~72particles containing ApoA-I and S-polypeptide had a much slower turnover rate than particles only containing S-polypeptide. On the basis of these results and arguments, it has been suggested that this isomorphic group of polypeptides be tentatively referred to as ApoI. Very recently, Harmony and her coworkers identified a protein associated with ApoA-I and cholesteryl ester transfer protein activity in some HDL subclasses. 75 This protein, termed ApoJ, was shown to be a unique component of HDL in human plasma consisting of two disulfidelinked nonidentical polypeptides designated ApoJct and A p o J f l 76 or, according to the ABC nomenclature, ApoJ-I and ApoJ-II. The apolipoprotein nature of this protein was confirmed by the isolation from HDL of its corresponding lipoprotein by immunoaffinity chromatography on an immunosorber with antibodies to ApoJ. 7s The possible occurrence of ApoJ in density classes other than HDL and VHDL has not yet been reported. Lipoprotein(a) (Lp(a)) first described as a genetic variant of LDL 4° represents a unique family of lipoprotein particles strongly associated with premature coronary artery disease. 7° The protein moiety of Lp(a) consists of ApoB covalently linked through a disulfide bond to a highly glycosylated, hydrophilic protein referred to as apolipoprotein a or Apo(a). 98'262The characteristic structural and antigenic properties of Lp(a) are due to Apo(a) which was shown to possess a high degree of homology to plasminogen (reviewed in 26°). Because Apo(a) belongs structurally to a superfamily of proteins including proteases of fibrinolytic and coagulation systems and does not appear to form its own family of lipoprotein particles or to possess a specific function in lipid transport, it is considered at the present time not to fulfill the criteria of an apolipoprotein. 26°Reports on the occurrence of additional minor apolipoproteins including the "proline-rich" polypeptide,224 the D-2 polypeptide TM and the "glycine-rich" polypeptide ~99are too preliminary to warrant their recognition as integral components of lipid transport. This brief survey of apolipoproteins included into the ABC system of nomenclature deserves a few additional comments. We have assumed in the initial design of this nomenclature that some apolipoproteins may consist of nonidentical polypeptides or

ApoA- and ApoB-containing lipoprotein families

111

subunits and, for this reason, introduced Roman numerals for their designation. This designation was applied to ApoA- and ApoC-polypeptides, because we considered for various reasons that ApoA-I and ApoA-II were, indeed, the constitutive polypeptides of ApoA (oe-protein) and ApoC-I, ApoC-II and ApoC-III constitutive polypeptides of ApoC. Although they occur very frequently clustered together in discrete lipoprotein particles, each of the ApoA- and ApoC-polypeptides may also be present in some lipoprotein particles as the sole polypeptide component of these two apolipoproteins. This applies, especially, to ApoA-I and ApoA-II polypeptides, both of which have been identified as sole protein constituents of lipoprotein particles called lipoprotein A-I (LP-A-I) and lipoprotein A-II (LP-A-II). Although very small amounts of lipoprotein C-I (LP-C-I), lipoprotein C-II (LP-C-II) and lipoprotein C-III (LP-C-III) have been identified among products of lipolytic degradation of triglyceride-rich lipoproteins, some of these lipoproteins may be ultracentrifugal artifacts. The designation of ApoA-IV was incorrect according to the principles of ABC nomenclature, because this apolipoprotein is not a constitutive polypeptide of ApoA nor does it occur very frequently as a cluster of ApoA-I/ApoA-II/ApoA-IV polypeptides; this designation was most probably prompted by the assumption that all apolipoproteins first identified in HDL ought to be considered as polypeptide constituents of ApoA, However, for practical reasons, this designation has been retained as originally proposed. In general, apolipoproteins listed in Table 1 from ApoA through ApoJ seem to fulfill most, if not all, of the criteria to be classified as integral protein components of lipid transport. Most apolipoproteins consist of several polymorphic forms which are designated according to the ABC nomenclature by Arabic numerals. In some cases, the polymorphism is genetically determined, while in other cases it arises from post-translational modifications. There are no generally established and accepted rules for naming polymorphic forms. A rigorous nomenclature proposed for phenotypes and genotypes of ApoE polymorphs 274 seems to have gained general acceptance. The three isoforms of ApoC-III are designated ApoC-III-0, ApoC-III- 1 and ApoC-III-2 according to the number of sialic TABLE 1. Nomenclature of Apolipoproteins* Apolipoproteins

ApoA

Constitutive polypeptides

ApoA-I ApoA-II ApoA-IV

ApoB

none

ApoC

ApoC-I ApoC-II ApoC-III

ApoD

none

ApoE

none

ApoF ApoG Aport ApoI

none none none none

ApoJ

ApoJ-I ApoJ-II

Polymorphic forms

ApoA-I_ ApoA-Io ApoA-I + i ApoA-II_l ApoA-II0 ApoA-II + i ApoA-IV _ l ApoA-IV 0 ApoA-IV+I ApoB- 100 ApoB-48, etc. ApoC-IIIo ApoC-|II I, etc. ApoDl ApoD 2, etc. ApoEi ApoE2, etc. Not known Not known Not known ApoIl ApoI2, etc. Not known

*Nomenclature for the polymorphic forms of ApoA-I, ApoAII and ApoA-IV was proposed by Sprecher e t al. u~ Arabic numeral 0 denotes the major mature forms of ApoApolypeptides.

112

P. AI~LU,OVlC

acid residues.~'49 Another proposal for the nomenclature of the polymorphic forms of apolipoproteins A-I, A-II, A-IV, C-I, C-II, D and H is based on their separation and identification by two-dimensional electrophoresis,m The mature isoform of an apolipoprotein is designated by numeral zero as, for example, ApoA-I0 or ApoA-II0. The other isoforms are coded by the number of negative or positive unit charges when compared to the mature form as, for example, ApoA-I+l, ApoA-I+2, ApoA-I_l, etc. The truncated forms of ApoB are named according to their apparent molecular weights or mobilities in SDS-polyacrylamide gel electrophoresis relative to that of intact ApoB designated as ApoB-100) 29 Studies on the physical and chemical properties of apolipoproteins culminated with the elucidation of the amino acid sequences of almost all apolipoproteins (A-I, A-II, A-IV, C-I, C-II, C-III, D, E and H) including that of ApoB, a single chain protein consisting of 4536 amino acid residues)9'ss'm'153 Because the purpose of this review is to present the evolving concepts regarding the classification and recognition of lipoprotein particles rather than the newly acquired knowledge about the structure of apolipoproteins, the reader is referred to several recent reviews concerned with this rapidly developing area of lipoprotein research.~5,53.16s,174a~9,2" In addition to their role in the formation and structural stability of lipoprotein particles, apolipoproteins perform crucial functions in the metabolic conversions of lipoproteins including their secretion, activation of lipolytic enzymes, retardation of their premature removal and recognition of their binding and removal sites on hepatic and extrahepatic cells.4S,TS,116,174,230,273

Studies on the chemical and metabolic properties of apolipoproteins have clearly demonstrated their significance as structural and functional determinants of lipoproteins and necessitated development of analytical procedures for their quantitative determination. Because of the complex chemical nature of lipoproteins, these procedures have been based almost exclusively on highly specific and sensitive immunological assays including radioimmunoassay, radial immunodiffusion, electroirnmunoassay, enzyme immunoassays, immunonephelometry and immunoturbidimetry.16'23'33'15°'164 None of these assays has been selected by a general consensus as a reference method, although it has been suggested that radioimmunoassay244 or enzyme-linked immunosorbent a s s a y 255 may be considered as candidates for such purpose. As pointed out by several authors, each of these immunoassays has certain advantages and limitations and will continue to be used as long as it meets specific needs of individual investigators. 16.23.33In discussing the advantages and disadvantages of various immunoassays and, especially, their suitability as the reference method, one should keep in mind the results of an international survey of ApoA-I and ApoB measurements which showed that the most significant source of error in measuring levels of these two apolipoproteins was the variability among laboratories rather than the variability among methods and antisera u s e d ) Is'244 However, despite the absence of an internationally accepted reference standard and considerable differences among laboratories regarding the values for normal concentration ranges of almost all apolipoproteins, it is posible within a laboratory to compare apolipoprotein levels of normolipidemic subjects with those of dyslipoproteinemic patients and draw some conclusions regarding the significance and usefulness of such measurements. In fact, the measurement of apolipoprotein concentration has already been shown in many instances to he more useful than that of lipids in diagnosing some dyslipoproteinemic states, predicting the presence or progression of coronary artery disease and providing clues about the chemical nature of lipoprotein particles. 16,3°'42,5°'t14a16,217 III. A P O L I P O P R O T E I N S

AS M A R K E R S

OF LIPOPROTEIN

PARTICLES

One of the main questions raised by the discovery and characterization of an unexpectedly large number of apolipoproteins was their distribution among lipoprotein density classes or electrophoretic bands. Another, equally important question was their localization on individual lipoprotein particles. The availability of highly purified apolipo-

ApoA- and ApoB-eontaining lipoprotein families

113

proteins permitted the preparation of monospecific, polyclonal antisera and their utilization as specific reagents for identifying and quantifying corresponding apolipoproteins. The initial application of immunodiffusion and immunodectrophoretic techniques, 2'4'15'"°,11:'t~ superseded later by quantitative immunoassays,~.5~3~5°demonstrated a wide distribution of most, if not all, apolipoproteins throughout the entire lipoprotein density spectrum of both normolipidemic and dyslipoproteinemic subjects. The protein constituents that have been identified in plasma chylomicrons are apotipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-III, E and H. 12'53'145'16s'211'261 In normolipidcmic subjects, the major apolipoproteins of VLDL are ApoB, ApoC-peptides and ApoE accounting for 40-50%, 25-40% and 10-15%, respectively, of the total apolipoprotein content;4,23,13°,147 the minor apolipoproteins of VLDL are ApoA-I, ApoA-II, ApoD, Aport and ApoI. 4'175'211'215The LDL contain ApoB as the major apolipoprotein (85-90% of the total apolipoprotein content) and ApoA-polypeptides, ApoC-polypeptides, ApoD, ApoE and ApoF as minor apolipoproteins. 4'23"137'147'154'156'215The ApoA-I and ApoA-II account for 75-85% of the total apolipoprotein content of HDL, but this lipoprotein density class also contains measurable quantities of ApoB, ApoC-polypeptides, ApoD, ApoE and A p o F 4'5's'23'147'25° as well as ApoA-IV, ApoG, Aport, ApoI and ApoJ.31,38,41,75,79,106,169,175,211,232,252,261,270The apolipoproteins identified in VHDL, frequently as ultracentrifugal artifacts, 14s include apolipoproteins A-I, A-II, A-IV, D, E, F, G, H, I and j.4.31,38,75,172,215It should be pointed out, that, even among normolipidemic subjects, there are noticeable variations and differences in the concentration and distribuiton of individual apolipoproteins depending on the age, sex and a number of genetic and environmental factorsJ 6'23 However, these variations, still within a relatively narrow range of values, are minimal in comparison with variations within and differences between various hypo- and hyperlipoproteinemic states ranging from a deficiency or absence to a several-fold increase in the concentration of a particular apolipoprotein(s). 16,23,33Qualitative and quantitative analyses of apolipoproteins showed clearly that each major lipoprotein density class or its subclasses contain more than a single apolipoprotein. All attempts at isolating a segment of the lipoprotein density spectrum that would contain a single apolipoprotein failed in achieving this goal. 3'8'15'112'147'154'156These findings had suggested that each lipoprotein density class contained a major apolipoprotein and a number of minor apolipoproteins, all of which were expected to be present on each lipeprotein particle of a specified density range. For example, all VLDL particles would have to contain ApoB as the major and ApoA-polypeptides, ApoC-polypeptides, ApoE, ApoD and Aport as the minor apolipoproteins. However, quantitative analyses excluded such structural arrangement of apolipoproteins by demonstrating that in each lipoprotein density class apolipoproteins occur in nonequimolar ratios. These results indicated an uneven localization of apolipoproteins on individual lipoprotein particles and suggested that lipoprotein density classes contain several types of lipoproteins of similar density properties but different apolipoprotein composition. Further studies on the localization and distribution of apolipoproteins on individual lipoprotein particles were aided substantially by the application of double diffusion analysis and crossed-immunoelectrophoresis. 2'4'8'12'15'31'84'182'25°Whole plasma or lipoprotein density classes were tested with various combinations of monospecific antisera to indiviual apolipoproteins and the corresponding immunodiffusion or immunoprecipitation patterns were interpreted according to the Ouchterlony's principles. 2°5 A nonidentity reaction between immunodiffusion lines or rockets indicated that any two apolipoproteins tested reside on separate lipoprotein particles, whereas an identity reaction indicated that such apolipoproteins are present on the same lipoprotein particle; partial identity between two apolipoproteins was taken as the evidence for the presence of two distinct lipoproteins, one of which contained a single and the other both apolipoproteins. Although not considered as the absolute criterion for structural integrity and identity of lipoprotein particles, these tests have provided crucial evidence that lipoprotein density classes contain several discrete lipoprotein particles rather than single, antigenically homogeneous, lipid-protein complexes. JPLR 30/2/~--B

114

P. ALAUPOVIC

The identification of several apolipoproteins and their localization on discrete lipoprotein particles of similar density properties but different apolipoprotein composition have revealed that major lipoprotein density classes are heterogeneous not only with respect to their hydrated densities, size, and lipid-protein composition but also with respect to their content and composition of distinct lipoprotein species. Although adding another dimension to the heterogeneity and complexity of plasma lipoproteins, apolipoproteins have emerged from these studies as the most useful markers for the identification and characterization of lipoprotein particles and for studying their interactions during all phases of lipid transport. IV. C H E M I C A L

C L A S S I F I C A T I O N OF P L A S M A L I P O P R O T E I N S CONCEPT OF LIPOPROTEIN FAMILIES

AND THE

The identification in major lipoprotein density classes of discrete lipoprotein particles of similar densities but different apolipoprotein composition demonstrated clearly that operationally defined lipoproteins cannot be considered as the fundamental chemical entities of this macromolecular system. Because apolipoproteins are the only chemically and immunochemically unique lipoprotein constituents, we have proposed some 25 years ago that apolipoproteins be used as markers for identifying individual lipoprotein particles and as the criterion for their classification,z3,t3 We have suggested initially that plasma lipoproteins consist of three lipoprotein families defined as "polydisperse systems of lipid-protein associations characterized by the presence of a single, distinct apolipoprotein or its constitutive polypeptides. ''3 These three lipoproteins were lipoprotein family A (LP-A) characterized by ApoA, lipoprotein family B (LP-B) by ApoB and lipoprotein family C (LP-C) by ApoC. It was thought that the predominance of LP-A in HDL, LP-B in LDL, and LP-C in VLDL reflected a certain degree of specificity in the lipid-binding capacity of each apolipoprotein. However, as polydisperse systems of particles, these lipoprotein families were found to be distributed through relatively wide segments of the density spectrum depending on the availability of lipid substrate and processes responsible for their degradation and removal. For example, LP-B particles have been detected mainly in the LDL2 subfraction (d = 1.019-1.063 g/ml) but, under certain metabolic conditions, substantial amounts of these particles can also be detected in LDL~ subfraction (d = 1.006-1.019g/ml) and VLDL. However, because they contain ApoB as the sole protein constituent, all these lipoprotein particles belong to the LP-B family. While recognizing the heterogeneity of lipoprotein families with respect to their density, size and lipid/protein composition, the chemical classification system provides for protein homogeneity and establishes apolipoproteins as the sole determinants of chemically definable entities. The overlap of various lipoprotein families along the density gradient appears to be one of the main reasons for the apolipoprotein heterogeneity of operationally defined lipoproteins. The early conceptualization of lipoprotein families had undergone several modifications necessitated by the discovery of additional apolipoproteins, improved immunochemical techniques and actual isolation and characterization of lipoprotein particles. However, despite these modifications pertaining mainly to the recognition and identification of discrete lipoprotein particles, the determinant role of apolipoproteins has remained as the major tenet of lipoprotein family concept. As mentioned earlier, LP-A, LP-B and LP-C families were considered to exist as separate polydisperse systems of lipoprotein particles extending and overlapping with one another in several segments of the density spectrum? This conclusion was based on their isolation as phospholipid-protein residues from VLDL, LDL and HDL and as intact lipoproteins from HDL. t4: The first isolation of intact lipoprotein families from HDL was accomplished by separation and removal of LP-B particles by immunoaffinity chromatography on an anti-ApoB immunosorber followed by fractionation of LP-A and LP-C particles by hydroxylaptite column chromatography)42 Application of this fractionation procedure to lipoproteins with d < 1.019 g/ml resulted in the isolation of products consisting of all three lipoprotein families. Because treatment of

ApoA- and ApoB-eontaininglipoprotein families

115

V L D L with monospecific antisera to A p o B or A p o C also resulted in a simultaneous precipitation of ApoA-, ApoB- and ApoC-containing lipoproteins, these findings suggested that, at higher densities,lipoprotein families LP-A, LP-B, and LP-C exist as separable entitiesand, at lower densities,as weak associations.3'4'Is5Further evidence for the existence of discretelipoproteinfamiliescontaining two or more apolipoproteins was provided by the resultsof double diffusionanalyses which showed that V L D L and LDL~ tested with various combinations of antisera to ApoA, ApoB and A p o C gave reactions of complete identity. 4,12,lS's4'155,16°:s°'2°7 These findingswere confirmed by actual isolationof lipoproteinparticlesthat contained severalapolipoprotcinsas integralconstituentsof their protein moieties. By using immunoaffinity chromatography with anti-ApoC immunosorbcr, Fcllin et al.s4 isolatedfrom a postprandial LDL~ subfraction lipoprotcinparticles which contained ApoA-peptidcs, ApoB and ApoC-pcptidcs as theirprotein constituents. Lee and Alaupovic 155 isolated from a LDL~ subfraction by immunoprecipitation with polyclonal antibodies to ApoB similar lipoprotein particlescharacterized by ApoB and ApoC-peptides as constituentsof theirprotein moiety. In a systematicstudy on lipoprotein particlesutilizingimmunosorbcrs with antibodiesto ApoB, A p o C and ApoE, McConathy et al. 4'1s4 showed that lipoproteins with d < 1.019 g/ml from normolipidemic subjects contained ca. 35% of lipoprotein particles with ApoB and ApoC-peptides, 45% of lipoprotein particles with ApoB, ApoC-peptides and ApoE, 5% of lipoprotein particles with ApoC-peptides and 20% of lipoprotein particles with unknown apolipoprotein composition. Application of this fractionation procedure to lipoproteins with d = 1.019-1.063 g/ml showed that this density class contained ca. 80% of lipoprotein particles with ApoB as the sole apolipoprotein, 14-15 % of lipoprotein particles with either ApoB and ApoC, or ApoB, ApoC and ApoE as protein constituents, and small amounts of lipoprotein particles with apolipoproteins A-I, A-II and D. 4as These results have demonstrated that, among lipoproteins with d < 1.063 g/ml (VLDL and LDL), some lipoprotein particles may contain a single apolipoprotein and some may contain several apolipoproteins regardless of their localization within a particular segment of the density spectrum. Similar findings were also reported in studies on HDL particles. Albers and Aladjem22 and Kostner et al) 43 were first to demonstrate that ApoA-I occurs in two subpopulations of HDL particles, one of which contains ApoA-I and ApoA-II and the other only ApoA-I as major protein moieties. These ApoA-I subpopulations of particles were shown to coexist in both HDL2 and HDL3 subclasses. 22'6~Results of early experiments with ultracentrifugally isolated lipoproteins suggested that, at higher densities, minor apolipoproteins such as ApoC, ApoD and ApoE occurred mainly as separate lipoprotein particles unassociated with one another or apolipoproteins A-I and A - I I . 2'3'112'146'1s1'2°°'25° However, the application of immunoaflinity chromatography to whole plasma or HDL preparations, isolated as infranatant fractions by a single preparative run at d = 1.063 g/ml, showed that substantial proportions of minor apolipoproteins were bound to lipoprotein particles characterized by ApoA-I or ApoA-I and ApoA-II as major protein c o n s t i t u e n t s . 4,s'6t'62,87:37,18'l'265These and previously described findings with ApoB-containing lipoprotein particles have been used as a basis for the present modified formulation of the lipoprotein family concept. The theory of lipoprotein families or particles is based on the proposition that apolipoproteins are the main determinants of their structural and functional properties. Accordingly, plasma lipoproteins are viewed as a mixture of discrete lipoprotein families defined by their apolipoprotein compositions rather than physical properties. Lipoprotein families that contain a single apolipoprotein are called simple lipoproteins, whereas lipoprotein families characterized by two or more apolipoproteins are called complex lipoproteins. 7 Simple and complex lipoprotein families are polydisperse systems of particles heterogeneous with respect to size, hydrated density and lipid/protein composition but homogeneous with respect to qualitative apolipoprotein composition. For example, lipoprotein particles that contain ApoB as their sole protein constituent may differ from one another in their physical characteristics and proportions of lipid and protein moieties but they all contain ApoB as the only identifiable apolipoprotein. Similarly, a complex

116

P. ALAUPOWC

lipoprotein family that contains apolipoproteins B, C-I, C-II and C-III represents a system of particles differing in their physical properties, lipid/protein ratios and percent composition of individual apolipoproteins; however, each particle of this lipoprotein family is characterized by the presence of the same four apolipoproteins. The nomenclature of lipoprotein families is simple and precise because it is derived from and based on the ABC nomenclature of apolipoproteins: lipoprotein families are named according to their qualitative apolipoprotein composition. Thus, a lipoprotein family that only contains ApoB is named lipoprotein B (LP-B); a complex lipoprotein family that consists of apolipoproteins B, C-I, C-II, and C-III is named lipoprotein B:C-I:C-II:C-III (LP-B:CI: C-II: C-III or, in abbreviated form, LP-B: C). If all three ApoC polypeptides are present in a lipoprotein family, they are referred to by the capital letter C; however, if only one or two of ApoC-polypeptides occur in a lipoprotein family, they should be identified by their Roman numerals. For example, lipoprotein particles having apolipoproteins B, C-I, C-II and C-III as their protein constituents are designated LP-B:C, whereas lipoprotein particles containing apolipoproteins B, C-I and C-II are called LP-B :C-I :C-II. Studies on the quantitative determination4'm6'23'33and distribution 4"5 of plasma apolipoproteins have demonstrated that apolipoproteins A (ApoA-I + ApoA-II) and B form two major groups of lipoprotein families, each of which consists of simple and complex lipoprotein particles. Apolipoproteins A-IV, C, D, E, F, G, H, I and J constitute the third minor group of lipoprotein families present mainly, but not exclusively, as simple lipoprotein particles. The general outline of plasma lipoproteins classified on the basis of their apolipoprotein composition is shown in Fig. 1. The following sections on the chemistry, function and clinical significance of lipoprotein families are not intended to represent a comprehensive review of all the available data of this growing field of inquiry but rather as a summary of recent accomplishments and an indication of future directions. V. F R A C T I O N A T I O N A N D C H A R A C T E R I Z A T I O N OF ApoA- A N D ApoB-CONTAINING LIPOPROTEIN FAMILIES

A. Introduction

ApoA- and ApoB-containing lipoproteins may be isolated by nonimmunological procedures including preparative ultracentrifugation, 74'1°3'1~5preparative electrophoresis on solid support, s3gel permeation chromatography, 22°and precipitation with anionic polyelectrolytes in the presence of divalent cations. 5: Although ApoA-containing lipoproteins may be separated from ApoB-containing lipoproteins by some of these procedures, they lack the specificity required for the separation of minor lipoproteins and fractionation of I. MAJOR LIPOPROTEINS 1.

2.

LIPOPROTEIN$ MNICH CONTAIN APOLIPOPROTEIN A (A-I + A - I I ) A.

SIMPLE LIPOPROTEIN$

B.

COMPLEXLIPOPROTEINS

LIPOPROTEINS WHICH CONTAIN APOLIPOPROTEIN B A.

SIHPLE LIPOPROTEINS

B.

COMPLEXLIPOPROTEIN$

I I . MINOR LIPOPROTEINS 1.

LIPOPROTEIN$WHICH CONTAIN ONE OF THE APOLIPOPROTEIN$ C. D. E. F. 6. H. I AND J A.

SIMPLE LIPOPROTEIN$

B.

COMPLEXLIPOPROTEIN$

FIG. 1. Classification of plasma Hpoproteins on the basis of apolipoprotcin composition.

ApoA- and ApoB-containing lipoprotein families

117

discrete lipoprotein families within each of the three lipoprotein groups (Fig. 1). The fractionation of lipoprotein families requires the application of highly specific immunologic procedures including immunoprecipitation and immunoaffinity chromatography or affinity chromatography on concanavalin A (ConA). Starting materials for the fractionation of lipoprotein families may be any type of lipoprotein preparations isolated by ultracentrifugation, preparative electrophoresis, gel permeation chromatography or precipitation. However, to avoid structural alterations of lipoproteins induced by some of these isolation procedures, 65's2J2°'126J4sit is recommended that whole plasma bc used as the most suitable starting material. A complete separation of ApoA- from ApoB-containing lipoproteins may be achieved by (a) immunoprecipitation with monospecific polyclonal antisera to ApoB, mg'72~:s(b) immunoaffinity chromatography with polyclonal anti-ApoB immunosorbers, :42'~44Js2 (c) immunoaffinity chromatography with "pan"-monoclonal antibodies to ApoB (monoclonal antibodies which bind equally to all ApoB-containing lipoprotein forms) used as a ligand, :36 and (d) affinity chromatography on ConAJ 8°'24° In all these procedures, ApoB-containing lipoprotein families are completely separated from ApoA-containing and minor lipoprotein families. Minor lipoprotein families can be separated from ApoAcontaining lipoprotein families by further fractionation on immunosorbers with antiApoA-I and anti-ApoA-II sera as shown in Fig. 2. Each of the four procedures for the separation of ApoA- and ApoB-containing lipoproteins has certain advantages and drawbacks and should be selected according to the needs and purposes of a particular investigation. The immunoprecipitation of ApoB-containing lipoproteins is a relatively simple procedure only requiring purified, polyclonal antisera to ApoB. Its advantages are the rapidity and capacity to precipitate relatively large amounts of ApoB-containing lipoproteins mainly dependent on the availability of purified antisera. However, because it results in the formation of insoluble ApoB-containing antigen-antibody precipitates, this procedure is more suitable for the isolation of larger quantities of ApoA-containing and minor lipoproteins than ApoB-containing lipoproteins. 2~5Although it is possible to recover antigenic components from insoluble antigen-antibody complexes, the rather cumbersome dissociation procedure usually results in relatively low yields of antigens, m The reverse immunoprecipitation, i.e. precipitation of ApoA-containing lipoproteins with a mixture of anti-ApoA-I and anti-ApoA-II sera is not recommended, because, under these experimental conditions, the LP-A-II: B : C: D : E particles would be coprecipitated with ApoA-containing lipoproteins and the soluble fraction would consist of minor lipoproteins and an incomplete complement of ApoB-containing lipoproteins. Immunoprecipitation with

~OLE e~S~ CoN A-UNRETAINED FRACTION ANTI-ApoA-II IHNUNOSORBER

lu

I R

LP-A-I MINOR LIPOPROTEINS

LP-A-I:A-II LP-A-II

ANTI-ApoA-I IHI4UNOSORBER

Iu LP-C LP-D LP-E LP-F, ETC.

1R LP-A-I

ANTZ-ApoA-I IHI4UNOSORBER

[u LP-A-II

1R LP-A-I:A-II

FIG. 2. Fractionation of ApoA-containing lipoprotein families from whole plasma or concanavalin A (ConA) unretained fraction by sequential immunoaflinity on immunosorbcrs with antisera to ApoA-I and ApoA-II. U -- unretained fraction, R = retained fraction.

118

P. ALAUPOXqC

anti-ApoA-I sera would circumvent the coprecipitation of LP-A-II: B: C: D: E particles but not the presence of LP-A-II particles in the soluble fraction. For these reasons, the use of anti-ApoB immunosorbers or affinity chromatography on ConA are the preferred procedures for the isolation of ApoB-containing lipoproteins. The important common feature of these procedures is that both the retained (ApoB-containing lipoproteins) and tmretained (ApoA-containing and minor lipoproteins) fractions are soluble and, thus, readily available for further fractionation. The advantage of affinity chromatography on CortA lies in its greater capacity to retain ApoB-containing lipoproteins than procedures based on immunosorbers with either polyclonal or "pan"-monoclonal antibodies to ApoB. The use of affinity chromatography on ConA for the separation of major and minor lipoprotein families is described in a separate contribution to these Proceedings.

B. Fractionation of ApoA-Containing Lipoprotein Families The most efficient procedure for the fractionation of ApoA-containing lipoproteins and minor lipoprotein families is based on the use of immunosorbers with either affinitypurified polyclonal antisera or ,'pan" monoclonal antibodies to ApoA-I and ApoAII. 4'8'10'27'35'39'62'65'73'87'124'131A85'197'249'251'265Several studies have shown that ApoA-containing lipoproteins consist of three lipeprotein families identified as LP-A-I, LP-A-I:A-II and L P - A - I I . sA0.22'26,27,35'39'61'62'73'124'I31'I43A76'I85A97,249'251These three lipoprotein families may be isolated either directly from whole plasma or from the unretained fraction generated by affinity chromatography of whole plasma on ConA (Fig. 2). Whole plasma or ConAtmretained fraction is first fractionated on an anti-ApoA-II immunosorber to separate LP-A-I and minor lipoprotein families from LP-A-II and LP-A-I:A-II. Further fractionation of the former, unretained fraction, on an anti-ApoA-I immunosorber results in the separation of LP-A-I and minor lipoprotein families, whereas the fractionation of the latter, retained fraction, yields LP-A-I:A-II and LP-A-II particles. It should be pointed out, however, that LP-A-II particles isolated directly from whole plasma will also contain LP-A-II:B:C:D:E particles (to be described in detail with other ApoB-containing lipoproteins) that can be removed by immunoaffinity chromatography on an anti-ApoB immunosorber or affinity chromatography on ConA. This step may be avoided by the use of ConA-unretained fraction as the starting material. The identification of minor lipoprotein families may be achieved by double diffusion analysis, t5 crossed immunoelectrophoresis 8 or two-dimensional electrophoresis 124'246followed, if deemed desirable, by actual isolation of lipoproteins with the use of specific immunosorbers. Preliminary results of studies on the characterization of LP-A-I and LP-A-I: A-II lipoprotein families have shown that each of these families consists of several subfamilies differing with respect to the content and composition of minor apolipoproteins 8'1°'39'62'72'73'87'124'137'184A97'265and the presence or absence of lecithin :cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein ( C E T P ) . 62'65'75'87'95 These subfamilies may be identified by various immunologic techniques s'15 and isolated by sequential immunoaffinity chromatography of LP-A-I, LP-A-I:A-II and LP-A-II on immunosorbers containing antibodies to minor apolipoproteins. Minor apolipoproteins found to be associated with ApoA-containing lipoproteins are apolipoproteins A-IV, C-I, C-II, C-III, D, E, F, H, I and J. However, systematic studies on the characterization and apolipoprotein composition of LP-A-I, LP-A-I:A-II and LP-A-II subfamilies have not been reported in the literature. Nevertheless, it is possible to make a few generalizations about the association of minor apolipoproteins, LCAT and CETP with ApoA-containing lipoprotein particles. Women have higher percentages of total plasma ApoC-peptides and ApoE in LP-A-I and LP-A-I:A-II families than men) 97 The LP-A-I:A-II family, regardless of sex and age, contains 70-90% of the total HDL content of apolipoproteins C-II, C-III, D and E. 39 Most of the plasma LCAT mass and CETP activity (ca. 70-80%) are associated with LP-A-I particles. 65's7 Finally, it should be realized that the majority (75-85%) of LP-A-I, LP-A-I:A-II and LP-A-II families do not contain minor apolipo-

ApoA- and ApoB-containinglipoproteinfamilies

119

proteins, sa°,39 The lipid and apolipoprotein composition of LP-A'I, LP-A-I:A-II and LP-A-II families has been reported by several investigators, s,~°.26,39,~2,73a24'137'~sS,~97,2s~ Although immunoaffinity chromatography has become the method of choice for the isolation and fractionation of LP-A-I and LP-A-I: A-II particles, their separation may also be achieved by agarose electrophoresis, m a combination of ion-exchange and hydroxylapatite column chromatography, m and chromatofocusing. 192.

C. Fractionation of ApoB-Containing Lipoprotein Families Development of the first procedure for the fractionation of ApoB-containing lipoprotein families was based on the results of double diffusion analysis, crossed immunoelectrophoresis and quantitative immunoassays of whole plasma and lipoprotein density classes suggesting that ApoB occurs in lipoprotein forms both as the sole protein and in association with ApoC-peptides and ApoE. 4'7,15,s4'ls4q56'la4,215 Because it was first designed for the fractionation of ApoB-containing lipoproteins in ultracentrifugally isolated VLDL, LDL~ and LDL2 preparations, this procedure, based on sequential immunoprecipitation of lipoproteins with purified monospecific polyclonal antisera to apolipoproteins E, C-III and C-II, consisted of two separate stages described in detail in several publications from this laboratory. 9'~9'2x'72'~55In the first stage, an aliquot of a lipoprotein density class was mixed with an equivalent amount of a polyclonal antiserum to ApoB and, after an incubation time of 2 hr at 4°C, the antigen-antibody complex was collected and removed. The soluble fraction was assayed for ApoC-peptides and ApoE to determine the amounts of these apolipoproteins unassociated with ApoB. In the next step, another aliquot of the same lipoprotein density class was mixed with an equivalent amount of a polyclonal antiserum to ApoE to precipitate all ApoE-containing lipoprotein particles. The amounts of precipitated ApoE and coprecipitated ApoC-peptides and ApoB were estimated as differences between the levels of these apolipoproteins in starting lipoprotein preparation and the soluble fraction remaining after the removal of precipitated lipoproteins. The precipitated lipoproteins consisted of LP-C-I : C-II: C-III: E (LP-B:C: E) and small amounts of LP-E; in some individuals this fraction also contained LP-B : E particles. The amount of LP-E was estimated by measuring the ApoE content of the soluble fraction after precipitation of ApoB-containing lipoproteins in the first stage of this procedure. The soluble fraction remaining after precipitation of ApoE-containing lipoproteins was treated with an equivalent amount of a polyclonal antiserum to ApoC-III and the ApoC-III-free soluble fraction was removed for further fractionation. The precipitated lipoprotein particles consisted of LP-B: C-I: C-II: C-III (LP-B: C) and small amounts of LP-C-III. The concentrations of these lipoprotein particles were determined as already described for ApoE-containing lipoproteins. If the soluble fraction remaining after precipitation of ApoC-III-containing lipoproteins contained some ApoC-I and/or ApoC-II, these lipoproteins were precipitated with anti-ApoC-I and/or anti-ApoC-II sera. However, because in the great majority of cases ApoC-I and ApoC-II had not been detected, the soluble fraction only consisted of LP-B particles. This procedure has been applied to the fractionation of ApoB-containing lipoproteins in VLDL, LDLI and LDL2 of normolipidemic subjects and patients with various dyslipoproteinemias. ~s'~9a64 Results of these studies have shown that LP-B is the major simple lipoprotein family and LP-B:C:E and LP-B :C the major complex lipoprotein families. The LP-B:E particles are a relatively minor ApoE-containing lipoprotein accounting in most subjects for less than 10% of the ApoE-containing lipoproteins present in VLDL, LDL~ and LDL2. It appears that LP-E, LP-C-I, LP-C-II and LP-C-III particles are mainly ultracentrifugal artifacts, because their concentrations in the unretained fraction isolated by affinity chromatography of whole plasma on ConA (Fig. 2) were found to be negligible in comparison with those estimated in lipoprotein density classes (unpublished results from this laboratory). Sequential immunoprecipitation is a useful procedure for identifying ApoB-containing lipoprotien families, for isolating larger quantities of LP-B particles or eliminating ApoE-containing lipoproteins. However, sequential immunoaffinity chromatography is the

120

P. ALAUPOWC

method of choice for isolating individual lipoprotein families, because both the retained and unretained fractions are recovered in soluble forms. Moreover, both polyclonal and monoclonal antibodies can be used for the preparation of immunosorbers. A study ~s2 on the optimal conditions for constructing and operating immunosorbers with polyclonal antibodies to ApoB has shown that immunosorbers with the highest capacity were obtained by cyanogen bromide activation of Sepharose and that among various dissociating agents tested, 3 M sodium thiocyanate was found to be the most effective desorbent for bound lipoproteins. To minimize the time of contact between lipoprotein particles and dissociating agent, it was found advantageous to construct immunosorbers with a protective layer of Sephadex G-25 placed below the immunosorber portion 72,]36,t37,1sl,ls2 where it serves as a desalting column by separating lipoproteins from the dissociating agent. By allowing lipoproteins only a brief contact with sodium thiocyanate, the Sephadex G-25 layer minimizes the potentially disruptive effect of this chaotropic agent on the structure of lipoproteins. In the same study, 182 the testing of the potentially nonspecific adsorption of lipoproteins was performed with albumin because of its capacity to bind fatty acids and lysolecithin. The almost quantitative recovery (97-100%) of albumin in unretained fractions showed that the nonspecific adsorption of a lipid-binding protein was minimal. The recovery of lipoprotein particles was found to be 80-90%. Several studies have clearly shown that, in contrast to ultracentrifugal techniques, immunoaffinity chromatography has no deleterious effect on the composition and metabolic properties of lipoproteins. 5s'62,64,65,88,]°°'124,136,155,ls2,lsS'265'275Moreover, these results have demonstrated that immunoaffinity chromatography is the mildest procedure for a reproducible isolation of lipoprotein particles of specific apolipoprotein composition. Its application to the fractionation of ApoB-containing lipoprotein families follows the already described sequential immunoprecipitation procedure with one important modification, i.e. the introduction of an anti-ApoA-II immunosorber as the initial step in the fractionatien scheme (Fig. 3). Previous studies using sequential immunoprecipitation of VLDL, LDLj and LDL2 have disclosed four major lipoprotein forms of ApoB including LP-B, LP-B: C, LP-B: C: E and LP-B:E. However, studies on the pathogenic mechanisms responsible for the impaired metabolism of triglyceride-ricb lipoproteins in Tangier disease have shown that Tangier

MHOLE PLASMA AFFINITY CHROMATOGRAPHY ON CONCANAVALINA [R APoB-CONTATNINGLIPOPROTEINS

L

lu APoA-CONTAININGLIPOPROTEINS MINOR LTPOPROTEINS ANTI-APoA-II INMUNOSORBER

[R LP-A-ZI:B:C:D:E (LP-A-ZZ: | COMPLEX) ANTI-ApoE INMUNOSORBER [R LP-B:E ANTI-APoC-III IMMUNOSORBER JR

U Lea

FIG.3. Fractionationof ApoB-containinglipoproteinfamiliesfromwholeplasmaby concanavalin A (ConA)affinitychromatographyand sequentialimmunoaffinitychromatographyon immunosorbers with antisera to ApoA-II, ApoE and ApoC-IlI. U ffiunretained fraction, R ffiretained fraction.

ApoA- and ApoB-containinglipoproteinfamilies

121

patients had significantly decreased activities of plasma postheparin lipoprotein lipase (LPL) and a lower reactivity of VLDL with human milk LPL. z69 It has also been established that ApoA-II content of Tangier VLDL was significantly higher than that of control VLDL suggesting a possible association between the abnormal apolipoprotein composition and low reactivity of triglyceride-rich lipoproteins. Fractionation of Tangier VLDL on an immunosorber with "pan'-monoclonal antibodies to ApoA-II revealed the presence of an additional ApoB-containing lipoprotein family characterized by apolipoproteins A-II, B, C-I, C-II, C-III, D and E as constituents of its protein moiety. ~4 This triglyceride-rich lipoprotein, named lipoprotein A-II: B: C: D: E (LP-A-II: B: C: D: E or LP-A-II: B-complex), accounted for 70-90% of the total ApoB content of Tangier VLDL. This polydisperse lipoprotein family has been shown to occur in varying concentrations in VLDL and LDL of normolipidemic and dyslipoproteinemic subjects. ~'~s As shown in Fig. 3, ApoB-containing lipoproteins separated from ApoA-containing lipoprotein by ConA affinity chromatography of whole plasma are first placed on an immunosorber with antibodies to ApoA-II to remove LP-A-II:B-complex. The unretained fraction is then concentrated and chromatographed on an anti-ApoE immunosorber to separate the remaining ApoE-containing lipoproteins LP-B:C:E and LP-B:E (retained fraction) from ApoE-free lipoprotein particles LP-B:C and LP-B (unretained fraction). The LP-B:C:E particles may be separated from LP-B: E particles on an anti-ApoC-III immunosorber. The separation of these two lipoprotein families may also be achieved by utilizing an immunosorber with monoclonal antibodies to ApoB which selectively bind to LP-B:E particles. TM In the final step of the fractionation procedure (Fig. 3), LP-B: C particles are separated from LP-B particles by chromatography on an anti-ApoC-III immunosorber. The construction of immunosorbers and their application to the fractionation of ApoBcontaining lipoproteins have been described in several reports from this and other laboratories. 14,21,58.72,84,88,100,134,136.144.182.272.275 At the present time, the major identified lipoprotein forms of ApoB are LP-B, LP-B:C, LP-B: E, LP-B: C: E and LP-A-II: B: C: D: E. Each of the ApoC-containing lipoprotein families may also contain small amounts of subfamilies characterized by the absence of one or two of the ApoC-peptides. It appears that these subfamilies represent products of a partial lipolytic degradation of their parent families or ultracentrifugal artifacts. A characteristic example is the LP-B:C-I:E subfamily identified as one of the in vitro generated products of LPL catalyzed degradation of LP-B:C:E particles. El The ApoB-containing lipoprotein families have several characteristic compositional features. They can be classified, for example, into ApoE-containing (LP-B:E, LP-B:C:E and LP-A-II : B-complex) and ApoE-free (LP-B and LP-B: C) lipoproteins or into ApoCcontaining (LP-B: C, LP-B : C: E and LP-A-II: B-complex) and ApoC-free (LP-B and LP-B:E) lipoproteins. The LP-B and LP-B:E families are characterized by cholesteryl esters as their main neutral lipid constituent, whereas LP-B: C, LP-B: C: E and LP-A-II: Bcomplex families contain triglycerides as their major neutral lipid component. It should be re-emphasized, however, that each lipoprotein family is a polydisperse system of particles differing in density, size and lipid/protein composition. As triglyceride-rich lipoproteins, LP-B: C, LP-B: C: E and LP-A-II: B-complex families have mainly density properties of VLDL and LDL~, whereas cholesteryl ester-rich LP-B and LP-B:E particles have density properties characteristic of LDL~ and LDL 2. However, the former lipoprotein families may also be present in the LDL2 and the latter lipoprotein families in VLDL and LDL~ density regions. In each lipoprotein family, the relative content of triglycerides decreases and those of cholesterol and cholesteryl esters increase with increasing density of lipoprotein particles (Table 2). This change in triglyceride/cholesterol and triglyceride/cholesteryl ester ratios is one of the important factors contributing to the polydispersity of lipoprotein particles. The other important factor is the changing weight ratio of total lipid to protein. The apolipoprotein composition of complex lipoprotein families also changes in a characteristic manner in that the relative content of ApoB increases and relative contents of ApoC-peptides and ApoE decrease with increasing density (Table 3). The LP-A-II:B-complex particles occurring in LDL density region have increased £PLR 30/2/~-C

122

P. ALAUI'OVIC TAet~ 2. Percent-Lipid Composition of ApoB-Containing Lipoprotein

Families in VLDL and LDL of Normolipidemic Subjects Density class

Triglycefides

Cholesterol

Cholesteryl

(%)

(%)

(%)

40.1 20.5

9.8 11.6

50.1 67.9

NDt 9.0

ND 17.9

ND 72.9

58.0 40.7

10.9 16.2

31.1 43.1

60.0 48.7

13.5 16.5

26.5 34.7

69.5 33.6

11.0 15.7

19.2 50.5

LP -B *

VLDL LDL L P - B :E

VLDL LDL L P - B :C

VLDL LDL LP-B:C:E

VLDL LDL L P - A -II : B

complex

VLDL LDL

*Results represent mean values of three separate samples of APOBcontaining lipoprotein families. t N D = not determined.

percentages of ApoA-II and ApoD in comparison with particles characterized by VLDL density properties. Preliminary results suggest that lipid and apolipoprotein composition of all major ApoB-containing lipoprotein families may differ within certain ranges between normolipidemic and dyslipoproteinemic subjects depending on the metabolic state of their lipid transport processes. VI. M E T A B O L I C A N D F U N C T I O N A L P R O P E R T I E S O F A p o A - A N D ApoB-CONTAINING LIPOPROTEIN FAMILIES

Results of several recent kinetic studies have demonstrated a marked metabolic heterogeneity of major lipoprotein density classes 190'191'236'256'272and suggested the presence of chemically distinct lipoprotein particles as the most probable cause for this newly r e c o g n i z e d heterogeneity, ss,163'236,272 Although studies on the metabolic and functional properties of discrete ApoA- and ApoB-centaining lipoprotein families are still in their early developmental phase, preliminary results suggest that lipoprotein particles characterized by specific apolipoprotein composition may also possess specific metabolic properties. TABLE 3. Percent Apolipoprotein Composition of Complex ApoB-Containing Lipoprotein

Families in VLDL and LDL of Hypertrigiyceridemic Subjects Apolipoproteins (%) Density classes

A-II

B

C-I

C-II

C-III

D

E

---

53.8 69.7

8.9 6.5

6.4 3.1

30.5 20.6

---

45.8 75.4

6.2 4.7

5.8 1.5

19.1 6.3

---

22.7 11.7

4.6 5.4

45.0 69.0

7.8 ND:~

6.3 1.0

20.3 6.7

0.9 5.9

14.4 11.9

L P - B :C*

VLDL LDL

-

-

1

m

m

LP-B:C:E

VLDL LDL L P - A - H :B

VLDL LDL

complext

*Results represent mean values of six separate samples of LP-B:C and LP-B:C:E families isolated from patients with phenotype V hyperlipoproteinemia. tResults represent mean values of three separate samples of LP-A-II :B-complex isolated from patients with Tangier disease) 4 :~ND ffi not determined.

ApoA- and ApoB-containing lipoprotein families

123

High density lipoproteins represent a complex mixture of LP-A-I, LP-A-I:A-II and LP-A-II, their subfamilies and minor lipoproteins. One of the first indications for a different metabolic behavior of LP-A-I and LP-A,I: A-II families was provided by studies showing that sterol effiux from cultured fibroblasts to human plasma was dependent on a complex H D L particle containing ApoA-I, ApoD and LCAT. sS's7It was suggested that LP-A-I:D lipoprotein family in association with LCAT and CETP may represent the functional complex responsible for esterifying and transferring cholesterol in plasma. 95 It appears that, in the absence or deficiency of LCAT activity, the maintenance of a balanced flux between cellular and plasma cholesterol is mediated by LP-E particles or an ApoE-enriched lipoprotein family of unidentified apolipoprotein composition, s6 The preferential association of LP-A-I particles with LCAT mass and LCAT and CETP activities was confirmed in a recent report indicating a minimal association of these latter activities with LP-A-I:A-II particles. 65 The importance of LP-A-I particles as a distinct metabolic entity was further supported by studies with cultured mouse adipose cells which showed that the cholesterol effiux from these cells was promoted by LP-A-I but not LP-A-I:A-II particles. 35 It was shown in the continuation of these experiments that the promotion of cholesterol efflux from adipose cells may also be mediated by LP-A-I:A-IV or LP-A-IV particles. 247 Although further studies are needed to identify the exact apolipoprotein composition of LP-A-I particles in promoting cholesterol efflux from peripheral cells, the antagonistic role of Apo-II or, more precisely, LP-A-I: A-II particles seems to be well documented by several investigators. 34's5'93'247A more detailed account of these important studies is presented in a separate contribution to these Proceedings. The binding to and uptake by HepG2 cells was reported to be significantly higher for LP-A-I than LP-A-I :A-II particles; 131it was concluded, however, that these results did not provide evidence for a specific role of ApoA-II in the binding and uptake of HDL-cholesteryl esters by HepG2 cells. The differential effect of nicotinic acid and probucol on LP-A-I and LP-A-I:A-II levels is another finding supporting the metabolic specificity and uniqueness of these two lipoprotein families; 27 this study has shown that nicotinic acid increases and probucol decreases the levels of LP-A-I particles without exerting any effect on the levels of LP-A-I: A-II particles. There are several reports in the literature suggesting that discrete ApoB-containing lipoprotein families with similar physical properties have distinct metabolic characteristics. The VLDL fraction retained on the heparin-Sepharose column was shown to have a higher ApoE/ApoC ratio than the unretained VLDL fraction; TM when injected into miniature pigs, the retained fraction was catabolized at a higher rate than the unretained fraction consistent with earlier findings that increased content of ApoE enhances while increased content of ApoC-III retards the uptake of triglyceride-rich lipoproteins by perfused rat livers. 23a'2a The fractionation of VLDL on an anti-ApoE immunosorber resulted in the separation of ApoE-containing and ApoE-free lipoprotein particles both of which contained ApoB-100. as However, the former lipoprotein particles bound to the LDL receptor on cultured human fibroblasts with a considerably higher affinity than the latter particles, suggesting the possible existence of two distinct catabolic pathways for ApoBcontaining lipoproteins. A similar finding has also been reported for LP-B and LP-B:E families isolated from LDL2 and shown to have significantly different binding characteristics for HepG2 cell membranes; J34 LP-B had a Kd value of 69 nM and LP-B:E a value of 21 riM. Taken together, these studies have shown that apolipoprotein composition and, more specifically, the presence or absence of ApoE has a significant effect on the catabolic fate of corresponding lipoprotein particles. Recently, we have monitored the in vitro lipolytic degradation of predominantly LP-B:C:E particles by measurement of ApoB-containing lipoprotein families. 2~ The dissociation of ApoC-peptides and ApoE was found to be proportional to the degree of triglyceride hydrolysis with LDL2 particles as the major and LDL1, H D L and VHDL particles as the minor lipoprotein density products of the LPL catalyzed lipolysis of VLDL (LP-B: C: E). After a 95% hydrolysis of triglycerides, plasma LDL2 consisted of 92.5% LP-B, 2.5% LP-B: C-I: E and 5% LP-B: C particles. These results have demonstrated that

124

P. ALAUI~VIC

the formation of cholesteryl ester-rich LP-B as the ultimate remnant of the in vitro lipolysis of triglyceride-rich LP-B: C: E particles only requires LPL as a catalyst and albumin as the fatty acid acceptor. However, under physiological conditions, other modulating agents are necessary to prevent the accumulation and regulate the interaction of phospholipid/cholesterol-rich ApoC and ApoE-containing particles released during the lipolytic degradation of triglyceride-rich lipoproteins. It is worthwhile mentioning that the lipoprotein family composition of LDL2 preparations, used as controls in these experiments, was very similar to that of in vitro generated LDL2 with 85-90% of LP-B, 5-10% of LP-B:C:E and 5% of LP-B: C as identified lipoprotein families. The rate of lipolytic degradation of triglyceride-rioh lipoproteins is not only dependent upon the level and activity of LPL but also on the reactivity of the tdglycedde-rich lipoprotein substrates with LPL. As part of our studies on the mechanism of hypertrigiyceridemia in Tangier disease, :69 we have established that Tangier VLDL are a less efficient substrate for human milk LPL than VLDL from normolipidemic subjects. As presented earlier, the apolipoprotein analyses revealed that Tangier VLDL had a significantly higher percentage of ApoA-II than normolipidemic VLDL. Further studies showed that increased relative contents of ApoA-II in Tangier VLDL were due to the presence of a newly recognized triglyceride-rich lipoprotein family named LP-A-II: B: C: D: E or LP-A-II: Bcomplex. 14This triglycedde-rich complex accounted for 80-90% of the total VLDL-ApoB, whereas LP-B:C: E and LP-B:C families constituted the remaining lipoproteins. The LP-A-II: B-complex was also detected in type V hyperlipoproteinemia and other dyslipoproteinemic states, albeit in concentrations lower than those detected in Tangier patients. The lipid and lipid/protein composition of LP-A-II: B complex were very similar to those of LP-B:C:E and LP-B:C families. ~4However, the measurement of the pseudo first-order rate constant (k~), defined as a measure of the reactivity of trigiyceride-rich lipoproteins with LPL, 269 was significantly lower for LP-A-II: B-complex (kl = 0.0148 + 0.002 min -I) than for a mixture of LP-B:C:E and LP-B:C (k, = 0.036 + 0.002 min-I). Because the k~ value of Tangier VLDL (kl = 0.017 min -~) was very similar to that of LP-A-II: B-complex, it was concluded that high levels of LP-A-II: B-complex are the most probable reason for the low substrate efficiency of Tangier VLDL. These findings have established a significant difference in the substrate reactivity towards LPL of major triglyceride-rich lipoprotein families and implicated the apolipoprotein composition as an important, if not the main, reason for this metabolic behavior. Very little is known about the mechanisms responsible for the formation of ApoA- and ApoB-containing lipoprotein families. Some ApoA- and ApoB-containing lipoprotein families may be formed both in the liver and intestine. However, it appears that ApoA-IV-containing particles are preferentially formed in the intestine and ApoE-containing lipoprotein particles in the liver. To gain some initial information on the hepatic ApoAand ApoB-containing lipoprotein families, we have studied the chemical composition of these lipoproteins in the medium of HepG2 cells. The ApoA-containing lipoproteins were isolated from HepG2 cell medium by affinity chromatography on ConA and separated by immunoaffinity chromatography on an anti-ApoA-II immunosorber into LP-A-I and LP-A-I:A-II lipoprotein families. 73 Approximately 47% of the total ApoA-I was present in LP-A-I and 53% in LP-A-I :A-II particles. Both ApoA families contained ApoE which accounted for 80% of the total ApoE in the medium; close to 60% of ApoE was associated with LP-A-I:A-II particles and 40% with LP-A-I particles. However, the percentage of LP-A-I and LP-A-I:A-II families unassociated with ApoE has not been determined. The possible association of ApoA-IV with LP-A-I and LP-A-I :A-II particles was tested by immunoblotting technique. However, ApoA-IV band was only identified in a fraction found to be free of LP-A-I and LP-A-I:A-II particles suggesting that ApoA-IV exists in the HepG2 cell medium as a separate LP-A-IV family. Both ApoA-containing lipoprotein families had a higher relative content of free cholesterol and a lower percentage of cholesteryl esters than the corresponding lipoprotein families from plasma. This difference in free cholesterol/cholesteryl ester ratio was most probably due to low activity of LCAT in the cell medium compared to that in plasma. '38 The ApoA families also had a higher

ApoA- and ApoB-containing lipoprotein families

125

content of ApoE but negligible amounts of ApoC-peptides compared to LP-A-I and LP-A-I:A-II particles from plasma, m,194;199 In an independent study, Chetmg et al. ~° confirmed and extended these findings by determining the shape and size of LP-A-I and LP-A-I:A-II particles. Both lipoprotein families consisted of discoidal and spherical particles with the former prevailing in LP-A-I:A-II. A study of size distributions clearly established the polydisperse character of both ApoA-I-containing lipoproteins; whereas 66-85% of LP-A-I :A-II particles had relatively high Stokes' diameters (9.2-17.0 nm), 74% of LP-A-I particles were located in the size region below 8.0 nm. Thus, the morphology and size distributions of LP-A-I and LP-A-I:A-II particles were found to be similar to those of nascent HDL ~13and HDL from patients with LCAT-deficiency.94 Results of these studies suggest that LP-A-I and LP-A-I :A-II families are independently formed in the liver, but must undergo substantial modifications in the plasma compartment to acquire structural and compositional properties of their plasma counterparts. The subfamilies of LP-A-I with ApoA-IV, ApoC-peptides, and Aport are most probably formed in the intestine. 1°7'~67'2H It appears that human colonic adenocarcinoma cell line Caco-2 may be used as a very convenient tool for studying the formation of intestinal lipoprotein families including the LP-A-I subfamilies. 122,25s Studies on the apolipoprotein composition of lipoproteins with d < 1.063 g/ml isolated from the culture medium of HepG2 cells showed that ApoB and ApoE were the major and ApoA-I and ApoA-II the minor apolipoproteins. 72'257To identify ApoB-containing lipoprotein families,72 the culture medium was chromatographed on an anti-ApoB immunosorber to separate the ApoB-containing lipoproteins from ApoA-containing lipoproteins. The fractionation of ApoB-containing lipoproteins on an anti-ApoE immunosorber resulted in the separation of LP-B and LP-B: E families. The content of ApoC-peptides was too small to be detected by immunoassays. In contrast to their counterparts in the plasma, both the LP-B and LP-B:E particles were characterized by high percentages of triglyceride (56-70%) and relatively low but similar percentages of free cholesterol (12-29%) and cholesteryl esters (15-22%) as neutral lipid components. Size distributions of spherical particles of LP-B and LP-B:E ranged between 100 to 500 A consistent with their presence in VLDL, LDL and HDL regions of the density spectrum. These two nascent hepatic ApoB-containing lipoprotein families differed from the corresponding plasma lipoproteins mainly with respect to the content of ApoC-peptides and the triglyceride/cholesteryl ester ratios. The possibility that triglyceride-rich LP-B and LP-B: E particles secreted by HepG2 cells might be the precursors of plasma LP-B : C and LP-B:C:E families remains to be tested in future experiments. The lipoprotein forms of ApoB formed in the intestine have not been identified. Studies on the characterization of lipoprotein families secreted by the HepG2 cells have shown that LP-A-I and LP-A-I :A-II families may be the major ApoA-containing and LP-B and LP-B:E the major ApoB-containing nascent hepatic lipoproteins. However, the mechanism and regulatory factors governing their intracellular assembly and extracellular modifications, as well as their possible interactions with nascent intestinal lipoproteins represent a challenging and fruitful area for future exploration. The present status of studies on the assembly of hepatic lipoproteins is presented in a separate contribution to these Proceedings.

VII. LIPOPROTEIN FAMILIES IN D Y S L I P O P R O T E I N E M I A S AND ATHEROSCLEROSIS A . Introduction

Due to the lack of a simple, reliable methodology for quantifying individual lipoprotein families, the data on the concentrations of ApoA- and ApoB-containing lipoproteins in normolipidemic subjects and patients with various dyslipoproteinemias are still sketchy or unavailable. However, despite this limitation, the measurement of lipoprotein families has already indicated the potential usefulness of their concentration profiles as a supplemen-

126

P. ALAUI'OVIC

tary diagnostic tool and a means for monitoring therapeutic intervention in dyslipoproteinemic patients.

B. Concentration Profiles of ApoA-Containing Lipoprotein Families So far, the measurement of LP-A-I and LP-A-I: A-II levels has been performed mainly in normolipidemic subjects by the use of four different procedures. These include the separation of LP-A-I and LP-A-I: A-II families by immunoaffinity chromatography on an anti-ApoA-II immunosorber followed by measurement of ApoA-I by either radial immunodiffusion 196'~97or electroimmunoassay; 39 the determination of LP-A-I and LP-AI:A-II by an enzyme-linked differential-antibody immunosorbent assay, the first immunoassay designed for the quantification of ApoA-containing lipoprotein families; 139 the measurement of LP-A-I particles by a differential electroimmunoassay on commercially available ready-to-use plates; 2°6 and precipitation of ApoA-II containing lipoprotein particles (LP-A-I:A-II) by an antiserum to ApoA-II and turbidimetric measurement of ApoA-I (LP-A-I) in the supernatant fraction) 77Although performed by the use of different methods and applied to various populations, the reported LP-A-I and LP-A-I:A-II concentrations in normolipidemic men and women seem to be within a reasonable range o f values. 27'39'62'69'139'177'196'197'206'214'245 Without exception, the concentrations of LP-A-I particles (reported in terms of ApoA-I levels) were found to be significantly higher in women (54.4-100 mg/dl) than in men (38.5-75.0 mg/dl). However, this sex-related difference has not been observed in the levels of LP-A-I:A-II particles which seem to be similar for women (75.0-97.3 mg/dl) and men (76.8-93.0 mg/dl). These results show that ca. 35-45% of plasma ApoA-I resides in LP-A-I and 55-60% in LP-A-I:A-II particles. However, in terms of total lipid and protein weights, 125 LP-A-I particles account for 20-28% and LP-A-I: A-II for 72-80% of the total mass of ApoA-containing lipoproteins. In a recent study 24s designed to determine the reference limits for LP-A-I levels in a presumably healthy population of about 1000 subjects of both sexes and age groups from 4 t o 7 0 years of age, the LP-A-I levels in 5th and 95th percentiles were 41 and 100 mg/dl for males and 40 and 122 mg/dl for females. The study indicated some variation in LP-A-I levels with age for both males and females. In males, the levels of LP-A-I reached highest values between ages 10-14 years and then, after a slight decline, remained constant. The LP-A-I concentrations increased steadily in females from childhood to the 55-70 year age group. Among various biological factors considered to have a possible effect on the levels of LP-A-I particles, the degree of overweight was the most important. Men and women who were overweight by more than 20% had slightly reduced levels of LP-A-I particles (11%) when compared to subjects of normal weight. There was a slight but statistically insignificant reduction in the LP-A-I levels in younger women (18-35 years of age) using oral contraceptives. Alcohol consumption had no significant effect on LP-A-I levels in men or in women. The concentration and composition of LP-A-II family of particles in normolipidemic subjects will be presented in a separate contribution to these Proceedings. In contrast to several studies on the concentration and composition of LP-A-I and LP-A-I: A-II families in normolipidemic, asymptomatic subjects, there are very few reports on the concentrations of these lipoprotein families in dyslipoproteinemic states. Ohta et al. have shown ~96that in young uremic girls maintained on continuous ambulatory peritoneal dialysis the levels of LP-A-I, but not LP-A-I:A-II, are significantly lower than in age-matched normal, asymptomatic subjects. However, young female patients with insulin-dependent diabetes mellitus had normal levels and composition of LP-A-I particles but differed from healthy, asymptomatic controls with respect to cholesterol, phospholipid and ApoC-III composition of LP-A-I : A-II particles.J98 The characterization of LP-A-I and LP-A-I:A-II families in subjects with ApoA-I-Milano variant showed that the levels of both lipoprotein families were proportionately reduced. 63The possible clinical significance of LP-A-I and LP-A-I: A-II levels in patients with coronary artery disease will be discussed in the final chapter of this review.

ApoA- and ApoB-containing lipoprotein families

127

C. Concentration Profiles of ApoB-Containing Lipoprotein Families At the present time, an accurate measurement of five major ApoB-containing lipoprotein families can only be achieved by sequential immunoprecipitation or sequential immunoaffinity chromatography of whole plasma or ApoB-containing density classes. 9'14,19'21 All concentration profiles to be presented in this review were determined by sequential immunoprecipitation of whole plasma or lipoprotein density classes and the results are expressed in terms of ApoB concentrations (mg/dl). Because these two procedures are too complex to be used for routine measurement of ApoB-containing lipoprotein families, simpler procedures have been developed for quantifying these complex lipoproteins. These procedures are based on the enzyme-linked differential-antibody immunosorbent assay originally developed by Koren et all 39 for quantitative determination of LP-A-I and LP-A-I:A-II particles. In its modified form, this assay has been applied to measurement of ApoB lipoproteins which contain ApoE and those which contain ApoC-III. a6'37,sg'~65The major disadvantage of this procedure is that it cannot differentiate clearly between these two types of ApoB-containing lipoproteins, because LP-B:C:E and LP-A-II:B-complex particles are measured both by anti-ApoE/anti-ApoB and Anti-ApoC-III/anti-ApoB assays. Another modification of the differential-antibody assay is utilizing a combination of "pan"-monoclonal antibodies to ApoB and a mixture of "pan"-monoclonal antibodies to ApoA-II, ApoC-III and ApoE to quantify LP-B particles and the mixture of all other complex lipoprotein families including LP-B: E, LP-B: C, LP-B: C: E and LP-AI I : B : C : D : E (LP-Bc); ~33in a number of clinical situations, the measurement of LP-B and LP-Bc particles may be adequate for evaluating the status of ApoB-containing lipoproteins. Studies on the fractionation of ApoB-containing lipoproteins have established that five major lipoprotein families occur in all normolipidemic, hypercholesterolemic and hypertriglyceridemic subjects studied. 4'm4'~6'ls'19As shown in Table 4, LP-B is the main ApoB-containing lipoprotein family in normolipidemic subjects. Approximately 94-95% of LP-B particles occur in LDL2. However, measurable levels of LP-B particles are also present in LDL~ and VLDL. The other two major ApoB-containing lipoprotein families are triglyceride-rich LP-B:C and "LP-B:C:E". Because in the initial studies on the fractionation of ApoB-containing lipoproteins it was not possible to differentiate between LP-B: E, LP-B: C: E and LP-A-II: B-complex particles, these three ApoE-containing lipoproteins were measured as a mixture of particles referred to as "LP-B:C:E". The LP-B:C and TABLE4. ApoB-Containing Lipoprotein Particles in Density Classes of Normolipidemic and Hyperlipoproteinemic Subjects* Lipoprotein particles

VLDL (mg/dl)

LDL~ (mg/dl).

LDL 2 (mg/dl)

Total (mg/dl)

1.0 (1.2)I" 1.4 (1.8) 2.1 (1.7)

3.8 (3.3) 0.4 (0.4) 1.9 (2.3)

69.6 (16.1) 5.0 (5.6) 8.8 (7.2)

74.4 6.8 12.8

4.0 (6.6) 1.4 (1.0) 3.0 (1.4)

12.2 (4.4) 2.4 (1.4) 3.5 (2.3)

201.0 (78.0) 19.3 (15.6) 42.0 (15.4)

217.2 23.1 48.5

0.5 (0.6) 38.7 (34.1) 24.4 (16.3)

6.2 (5.7) 4.8 (5.3) 3.3 (1.9)

42.1 (24.4) 9.6 (5.6) 14.0 (13.0)

48.4 53.1 41.7

Normolipidemia (n = 15) LP-B LP-B:C "LP-B:C:E"

Familial hypercholesterolemia heterozygotes

(n 3) =

LP-B LP-B:C "LP-B:C:E"

Hypertriglyceridemia phenotype IV

(,=4)

LP-B LP-B:C "LP-B:C:E"

*Determination of LP-B and "LP-B:C:E" particles was performed by sequential immunoprecipitation;19results are expressed in terms of ApoB content (mg/dl) of corresponding particles. LP-B:C:E particles consist of all particles which contain ApoE, i.e. LP-B:E, LP-B:C:E and LP-A-II:B:C:D:E. tMeans (SD).

128

P. AL^Ur~VIC

"LP-B:C:E" particles account, on an average, for almost 80% of ApoB lipoproteins in VLDL and 40% in LDL~. A relatively high percentage (16%) of these partially delipidized triglyceride-rich lipoproteins may be detected in LDL2 confirming our previous data on the ApoC-III and ApoE contents of LDL: ms~ and demonstrating marked lipoprotein heterogeneity of this density class. The increasing percentage of LP-B particles and decreasing percentages of LP-B: C and "LP-B: C: E" particles in VLDL, LDLm and LDL2 mainly result from the lipolytic degradation of triglyceride-rich LP-B: C and "LP-B: C: E" particles and the formation of cholesteryl ester-rich LP-B as the final product of this catabolic process. 2m Differences in the concentrations of ApoB-containing lipoproteins between patients with heterozygous familial hypercholesterolemia and primary hypertriglyceridemia (phenotype IV) 'reflect characteristic alterations in the metabolism of these lipoproteins (Table 4). In comparison with normolipidemic subjects, hypercholesterolemic patients have very high levels of LP-B particles in all density classes, but, especially, in LDL2. They also have relatively high levels of LP-B: C and "LP-B: C: E" particles in LDL~. The LP-B: E particles account for the major part of the latter lipoprotein group. This lipoprotein profile of hypercholesterolemic patients, also seen in homozygous patients with familial hypercholesterolemia and in patients with phenotype IIA, reflects the impaired uptake of LP-B and, possibly, LP-B:E and LP-B:C particles. In contrast to normolipidemic and hypercholesterolemic subjects, the lipoprotein profile of hypertriglyceridemic patients is characterized by high concentrations of LP-B:C and "LP-B:C:E" particles in VLDL and low concentrations of LP-B in LDL:; the levels of LP-B: C and "LP-B: C: E" are relatively low in both LDLI and LDL2. The accumulation of these two lipoproteins in VLDL reflects their simultaneous overproduction and decreased lipolytic degradation. These results suggest that the differences between normal and dyslipoproteinemic states result mainly from quantitative rather than qualitative composition and distribution of major ApoB-containing lipoprotein families. Hypercholesterolemic states are characterized by increased concentrations of LP-B particles, and the hypertriglyceridemic states by increased concentrations of LP-B: C, LP-B: C: E and, as recently demonstrated, LP-AII:B-complex particles. 14'm8Preliminary results suggest that the lipoprotein family profiles of other primary and secondary hyperlipoproteinemias correspond more closely to either hypercholesterolemic or hypertriglyceridemic lipoprotein particle profiles, depending most probably on the nature of the underlying metabolic impairment of lipid transport, msThe variations in the concentrations of LP-B, LP-B:C and "LP-B:C:E" among normolipidemic or dyslipoproteinemic subjects of a phenotypically or genotypically defined subpopulation suggest that lipoprotein particle profiles reflect not only a specific metabolic impairment characteristic of a given patient subpopulation but also the degree of such a defect among individual subjects. The measurement of the concentration profiles of ApoB-containing lipoprotein families in larger population samples and studies on various endogenous and exogenous factors that may affect these concentrations will depend to a large extent on future development of simpler procedures for quantitative determination of this clinically important group of plasma lipoproteins. D. Lipoprotein Families and Coronary Artery Disease

Results of many epidemiologic and clinical studies have demonstrated that impairments in metabolism of plasma lipoproteins are one of the most important risk factors for the genesis and development of coronary artery disease. ~6~ These abnormalities include increased levels of VLDL and LDL and decreased levels of HDL. Most epidemiologic studies have concluded that hypercholesterolemia is more significantly associated with coronary artery disease than hypertriglyceridemia and that cholesteryl ester-rich LDL particles have a greater atherogenic potential than triglyceride-rich VLDL particles. It appears, however, that in some subsets of the population plasma concentrations of triglycerides are a more accurate predictor of coronary heart disease and mortality that

ApoA- and ApoB-containinglipoproteinfamilies

129

concentrations of cholesterol. 54'ss'57'151 In general, hypertriglyceddemia with or without associated hypercholesterolemia occurs more frequently in patients with premature coronary artery disease than hypercholesterolemia.TM Impaired lipolytic degradation of triglyceride-rich lipoproteins of intestinal and hepatic origin leads to the formation of remnant lipoprotein particles only partially depleted of triglycerides but enriched in cholesterol. ~28 Several clinicalu2'253 and metabolic 92'99'276 studies have suggested that these modified triglyceride-rich lipoproteins may have atherogenic potential similar, if not equal, to that of typical cholesterol-rich LDL. Because ApoB is the characteristic constituent of chylomicrons, VLDL and LDL particles, it has become customary to use ApoB as a marker of potentially atherogenic lipoproteins. Although the exact chemical properties of atherogenic lipoprotein particles have not been established, it is generally considered that ApoB-containing lipoproteins differ in their atherogenic potential which seems to increase with decreasing size and increasing density of lipoprotein particles. It appears that the optimal atherogenic size and lipid and protein composition are expressed most prominently in lipoprotein particles of intermediate density (d--1.006--1.040 g/ml). 99'187'194 If supported by additional evidence, the concept of relative atherogenicity of ApoB-containing lipoprotein particles may be of considerable theoretical and clinical significance. The identification of five major ApoB-containing lipoprotein families differing in their apolipoprotein composition has not only revealed the complex chemical nature of this lipoprotein group but also raised the question of their relative atherogenic potential. In the absence of a direct method for determining the relative atherogenicity of lipoprotein particles, we have based our preliminary investigation of this important problem on data derived from the CLAS study (Cholesterol-Lowering Atherosclerosis Study)42'43 and separate studies of a group of patients with coronary artery disease m and a group of patients with noninsulin dependent (type II) diabetes mellitus." The CLAS study was a randomized, placebo-controlled, angiographic trial involving 162 nonsmoking men with progressive atherosclerosis and previous coronary bypass surgery who had been treated with combined colestipol and niacin therapy for 2 years.43 The treatment reduced the progression of existing lesions and induced regression in some lesions; there were 61% nonprogressors in the drug group and 40% in the placebo group. All patients were characterized by measurements of serum lipids, lipoprotein lipids and apolipoproteins A-I, B and C-III. In addition, ApoC-III was determined in heparin-Mn 2+ supernates ( " H D L ' ) and precipitates ("VLDL + LDL") and the ApoC-III-supernate/ ApoC-III-precipitate ratio was used as a measure of the reciprocal relationship between VLDL and HDL particles or the efficiency of processes responsible for the degradation of triglyceride-rich lipoproteins. 6 The univariate analysis of risk factors comparing coronary progressors and nonprogressors showed that, in the placebo group, progressors had higher levels of serum cholesterol, non-HDL-cholesterol, triglycerides, LDL-cholesterol, ApoB and ApoC-III than nonprogressors. 42 In the drug group, progressors had significantly lower levels of ApoC-III-supernate as the only on-trial predictor of the coronary progression. The multivariate analysis showed that non-HDL-cholesterol levels were the significant independent predictor of coronary progression in the placebo group and ApoC-III-supernate levels in the drug group. These results have demonstrated the importance of triglyceride-rich lipoproteins in the progression of atherosclerotic lesions; however, they also showed that drug treatment had a significant effect on the levels of LDL-cholesterol and ApoB and that the higher levels of ApoC-III-supernate in the absence of drug therapy were not protective. The measurement of ApoB-containing lipoproteins in a limited number of drug-treated patients showed a highly uniform and statistically significant reduction of LP-B levels but no effect on the levels of LP-B: C and "LP-B:C:E" particles. TM These results suggested that LP-B particles had a significant atherogenic potential as evidenced by increased number of nonprogressors in the drug group and progressors in the placebo group. However, patients who also had increased levels of LP-B: C and "LP-B: C: E" particles, as indicated by decreased levels of ApoC-IIIsupernate, showed progression of atherosclerotic lesions despite reduced levels of LP-B particles. It seems, therefore, that intact or partially delipidized triglyceride-rich LP-B:C

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and "LP-B: C: E" particles (LP-B: C: E or LP-A-II: B-complex) also carry a certain risk for coronary artery disease similar to that of LP-B particles. These results also demonstrated a selective effect of nicotinic acid and colestipol combination on ApoB-containing lipoproteins. The results of Helsinki Heart Study m may be interpreted as an additional support for the significance of triglyceride-rich lipoproteins as a risk factor for premature coronary artery disease. A randomized treatment of dyslipoproteinemic men with gemfibrozil for 5 years resulted in a 34% reduction in the incidence of coronary heart disease accompanied by modest decreases in the levels of serum cholesterol (10%) and LDL-cholesterol (11%), a marked decrease in triglyceride (35%) and a moderate increase in HDL-cholesterol (11%). These results were interpreted as a further confirmation of the importance of LDL levels as a risk factor and H D L as a protective factor for coronary artery disease. However, despite minimal decreases in LDL-cholesterol levels, patients with phenotypes IIB and IV had greater reduction in incidence of coronary end points than patients with phenotype IIA. A study on the effect of gemfibrozil on ApoB-containing lipoprotein families in patients with phenotype V ~ and phenotype IV (unpublished results) showed a significant reduction in the levels of LP-B:C and "LP-B:C:E" particles but no effect on the levels of LP-B particles. This finding has provided, at least, a partial explanation for the relatively modest effect of gemfibrozil on patients with phenotype IIA whose high levels of LP-B particles had not been reduced sufficiently to produce the same favorable effect achieved with reduction of LP-B: C and "LP-B: C: E" levels in phenotypes IIB and IV. In addition, this finding represents another case of a selective effect of a hypolipidemic drug on ApoB-containing lipoprotein families. In a recent study 135 of 67 normotensive, nondiabetic patients with angiographically documented coronary artery disease and normal or moderately increased levels of total cholesterol (215 + 36 mg/dl) and triglycerides (143 + 86 mg/dl), significant correlations were observed between the severity of atherosclerotic lesions and ApoC-III in heparin precipitates, HDL-cholesterol, triglycerides, ApoE and ApoB, but not total cholesterol or LDL-cholesterol. A positive correlation was also observed between coronary artery disease scores and levels of triglyceride-rich LP-Bc particles directly measured by an enzyme immunoassay. Thus, in this patient population, the constituents of triglyceride-rich lipoproteins appeared to be better predictors of the severity of atherosclerotic lesions than total cholesterol or LDL-cholesterol. Results of these clinical trials suggest that both the cholesteryl ester-rich LP-B particles and triglyceride-rich LP-B: C, LP-B: C: E and LP-A-II: B-complex particles contribute to the development and progression of atherosclerotic lesions. Based on circumstantial evidence, the atherogenicity of LP-B particles seems to be greater than those of other ApoB-containing lipoprotein families. The most compelling argument for this conclusion is the association of very high levels of LP-B '6 with the fulminating coronary heart disease in homozygous patients with familial hypercholesterolemia. Preliminary studies on the relative atherogenic potential of triglyceride-rich ApoBcontaining lipoprotein particles have shown that, in VLDL of patients with noninsulin dependent (type II) diabetes mellitus, t~ there was a highly significant increase in the levels of LP-B:C (11.3 vs 1.2mg/dl, p