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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3542-3556.)
© 1997 American Heart Association, Inc.

Articles

Lipoprotein Heterogeneity and Apolipoprotein B Metabolism

Chris J. Packard; ; James Shepherd

From the Institute of Biochemistry, Glasgow Royal Infirmary, Glasgow, G4 OSF, UK.

Correspondence to Professor Chris J. Packard, Department of Pathological Biochemistry, 4th Floor, Queen Elizabeth Building, Alexandra Parade, Glasgow G31 2ER UK.

	Abstract

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
Abstract The apolipoprotein B containing lipoproteins VLDL, IDL, and LDL exhibit variation in their structure, function, and metabolism. These major lipoprotein classes can be fractionated into apparently discrete components by density gradient centrifugation or affinity chromatography. Examination of the behavior of subfractions in vivo reveals the presence of metabolic channels within the VLDL-LDL delipidation cascade so that the pedigree of a lipoprotein in part determines its metabolic fate. Evidence from VLDL and LDL apoB turnovers together with epidemiological data allows the construction of a quantitative model for the generation of small, dense LDL. This lipoprotein subspecies is one component of the dyslipidemic syndrome known as the atherogenic lipoprotein phenotype, a common disorder in those at risk for coronary heart disease. Understanding lipoprotein heterogeneity is an essential step in the further discovery of the pathogenesis of atherosclerosis and in the tailoring of pharmacologic treatment for subjects at risk.

Key Words: kinetics • VLDL • plasma triglyceride • small, dense LDL • lipoprotein lipase • hepatic lipase

	Introduction

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
Classical studies by Gofman and his colleagues¹ in the 1950s and 1960s using the analytical ultracentrifuge, revealed for the first time the heterogeneous nature of the complexes in plasma that were responsible for lipid transport. Peaks and troughs in the Schlieren pattern were clearly observed in the S_f 0 to 400 range and the same was true of high density lipoproteins. However, lipoproteins cannot be isolated from an analytical ultracentrifuge and the publication² of a convenient method for preparing total lipoprotein classes focused attention on the broad properties of very low, low, and high density lipoproteins. The structure, function, and metabolism of these lipoprotein classes has been intensively investigated over the last three decades but until recently little work had been done to discover if the underlying heterogeneity is important in determining the functional properties of a lipoprotein, in particular its propensity to promote or inhibit the processes that lead to atherosclerosis. Recognition that subfractions of lipoproteins may have individual significance in terms of association with disease came first for HDL. Division into HDL₂ and HDL₃ revealed that the former exhibited a strong, negative relationship with risk of CHD and was the component of the spectrum that generated most of the variation in total HDL plasma concentration.³ Within the S_f 0 to 400 range of apolipoprotein B containing particles it was established that IDL of density 1.006 g/ml to 1.019 g/ml should be considered a separate major class with distinct metabolic properties, but apart from sporadic reports there was no comprehensive evaluation of the role of subfractions within the VLDL-LDL range. The view of the majority of workers in the field in the 1970s and early 1980s (the authors of this review included) was that within a density interval, the spectrum of lipoprotein particles differed only slightly from one another in lipid content or apoprotein composition and there were no step changes or discrete entities, ie, lipoprotein classes were polydisperse. However, in later years when we or others isolated subfractions of VLDL or LDL and studied their properties in vivo or in vitro it became increasingly obvious that this paradigm was incorrect; the behavior of one component could differ markedly from that of another.^{4 5} In a similar vein, in a series of seminal studies it was demonstrated that lipoprotein components of discrete size were present within LDL and one of these, the smallest species of 240 Å to 250 Å diameter, was associated with CHD risk in a way that particles of larger size were not.^{6 7} Thus, it is now widely accepted that lipoprotein classes are paucidisperse, ie, composed of a limited number of components with distinct properties and we are only just beginning to uncover the basis for this heterogeneity and the consequences for disease. Parenthetically, it should be noted that some investigators, notably Alaupovic et al,⁸ have continually made the case for regarding lipoprotein classes as mixtures of discrete components.

Heterogeneity within lipoprotein classes can be the result of differing lipid content, different apoprotein composition, altered protein conformation or as yet unidentified structural variation (eg, carbohydrate content). Since VLDL, IDL, and LDL are linked in a continuous metabolic cascade in which lipid (ie, mainly triglyceride) is lost in a series of small lipolytic steps, the delipidation process itself cannot give rise to discrete subfractions. They must arise either by the insertion of new material within a narrow density interval or by the formation of an initial stable product that must undergo a step change to the next state. Immunological methods are used to isolate fractions of differing apoprotein content from within a lipoprotein class and a sometimes bewildering array of subfractions containing various combinations of apoproteins can be prepared by immunoaffinity chromatography.^{8 9} It is unclear as to how many of these species represent entities that could be considered discrete subfractions, since all apoproteins except apoB are known to exchange between particles. Evidence is emerging, however, that specific fractions of different apoprotein composition may retain their identity long enough in plasma for the properties of the lipoprotein to be modified. Again, the most convincing data in this regard have come from studies of HDL particles that contain either apo-AI and no apo-AII(Lp-AI) or both main apo-A proteins (Lp-AI/AII). Kinetic investigations have revealed that these species have individual turnover rates.¹⁰

The following review focuses on heterogeneity in the apoB-containing lipoprotein classes and interprets kinetic studies of apoB metabolism in light of underlying structural and functional variation. This is not meant to be an esoteric exercise, rather it arises from the conviction that only a limited amount of information about the plasma lipid transport system can be gained from examination of a whole lipoprotein class, if indeed the true building blocks of the system are its component subfractions. Furthermore, at a time when the use of lipid lowering agents in both primary and secondary prevention of CHD is now accepted medical practice, it is essential that drugs are targeted towards those at highest risk for both ethical and financial reasons. Understanding in detail the aberrations in lipid metabolism and lipoprotein substructure that give rise to a propensity to develop atherosclerosis will help identify those measurements that can be made to assist in prognosis beyond the assay of total plasma cholesterol or the LDL to HDL ratio.

	Structure and Function of VLDL Subfractions

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
VLDL comprises the class of lipoproteins present after an overnight fast that float when plasma is subjected to a centrifugal force. On the basis of this broad definition there is no reason why this lipoprotein fraction should be homogeneous and indeed any investigation of its structure has revealed marked heterogeneity in size and in lipid and apoprotein composition. VLDL is synthesized and secreted continuously by the liver. Lipoprotein assembly is initiated by the addition of lipid to the growing apoB chain in the rough endoplasmic reticulum. More lipid, principally triglyceride, is added to nascent VLDL particles as they pass along the secretory pathway. Details of the VLDL assembly process are beyond the scope of the present review but have been described in a number of recent articles.^{11 12 13} However, concepts derived from cell culture studies will be revisited following a discussion of the in vivo properties of VLDL subfractions and what these reveal about the synthetic machinery.

Two methods have been employed most commonly to fractionate VLDL into structurally and metabolically discrete components. These are centrifugation that separates mainly on the basis of size and density (ie, particle lipid content) and affinity chromatography where differences in apoprotein composition or conformation are exploited. VLDL vary in size from about 350Å to 700Å diameter, which corresponds to 8-fold range in volume, ie, from 22x10⁶ ų to 180x10⁶ ų . Most of the difference in size is due to the triglyceride core since the phospholipid/apoprotein coat is of constant thickness in lipoproteins.^{14 15} Separation by density is likely to be a useful approach since the subfractions generated vary in triglyceride content and will have arisen either because lipoproteins with a different lipid load have been released by the liver or because lipolysis has produced a smaller particle from a larger one. In fact, there is evidence that both processes are responsible for the heterogeneity observed. Techniques such as gel chromatography or density gradient centrifugation have divided VLDL into multiple fractions^{14 15 16 17} but a convenient, commonly used and highly reproducible method is that of cumulative flotation centrifugation,¹⁸ which provides two or three subfractions in a concentrated form suitable for further analysis, namely VLDL₁ of S_f 60 to 400 and VLDL₂ of S_f 20 to 60 or alternatively VLDL₁ of S_f 100 to 400, VLDL₂ of S_f 60 to 100 and VLDL₃ of S_f 20 to 60. Compared to larger VLDL, VLDL of S_f 20 to 60 are smaller, enriched in cholesteryl ester, deplete in triglyceride and have a lower ratio of apoE and apoC to apoB.^{14 15}

	Properties of VLDL Subfractions Separated by Density

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
In a series of studies we investigated the metabolism of apoB in VLDL subfractions in normal and dyslipidemic subjects.^{5 19 20 21} The results, summarized diagrammatically in Fig 1, revealed not only differences in the kinetics of the component fractions but also the presence of "metabolic" channeling within the VLDL-LDL delipidation cascade where apparently parallel (ie, nonintersecting) processing pathways generate IDL and LDL products of differing metabolic potential from precursors in the VLDL range. In early experiments we trace-labeled VLDL of S_f 100 to 400 with the idea of following the various lipolytic and catabolic steps through to LDL, in line with current models at the time we envisaged a single delipidation cascade from the largest VLDL particles to LDL. However, on injection of radioiodinated S_f 100 to 400 VLDL into subjects we observed that while substantial amounts of its apoB appeared in smaller VLDL and IDL following delipidation of the particle, less than 10% was converted to LDL.¹⁹ Thus, large VLDL was postulated not to form LDL to a significant degree, a tenet that has been confirmed by others.²² Rather, its delipidation ceases in the VLDL or IDL density ranges where it is thought to generate remnants that persist in the circulation for considerable periods (Fig 1). Intermediate sized VLDL of S_f 60 to 100 has kinetic properties similar to those of S_f 100 to 400 VLDL, although more of the apoB from this interval is delipidated to LDL.^{5 19 20} ApoB containing particles in the S_f 20 to 60 density range, in contrast, are rapidly and efficiently converted to LDL with >50% of the tracer being observed in LDL within 24 hours after injection.¹⁹ As explained in detail elsewhere,²³ analysis of dual tracer turnovers using ¹³¹I-VLDL S_f 60 to 400 and ¹²⁵I-VLDL S_f 20 to 60 by multicompartmental modeling led to a complex but physiologically important conceptualization of apoB metabolism that placed kinetic heterogeneity at the center of an understanding of how VLDL and LDL synthesis and catabolism are regulated. Studies in a wide variety of subjects indicate that the liver synthesizes and secretes VLDL particles that range in size and density across the full S_f 20 to 400 spectrum. Furthermore, there is increasing evidence that the production of VLDL₁ (S_f 60 to 400) and VLDL₂ (S_f 20 to 60) are regulated independently of one another.^{21 24 25} VLDL₁ production was increased in subjects with high-normal compared to low-normal plasma triglyceride levels [C.J.P. et al, 1997, unpublished], was stimulated by estrogen treatment in post-menopausal females,²⁴ and specifically inhibited by the infusion of insulin to normolipidemic men.²⁵ VLDL₂ production was not perturbed by the administration of these hormones but was found to be elevated in moderately hypercholesterolemic subjects.²¹ Increased LDL production has long been considered to be a major cause of raised LDL levels in common forms of hypercholesterolemia²⁶ and our investigations located enhanced hepatic output into VLDL₂ as the likely source of this metabolic abnormality (Fig 1). The mechanism by which the liver is able to vary the amount of large versus small VLDL secreted is unknown. Recent experiments in cell culture^{12 13} suggest that VLDL assembly is more complex than was originally thought. A two-step process is envisaged in which a small lipoprotein particle containing at first little triglyceride is formed in the rough ER and then the bulk of the triglyceride core added to this at the junction of the rough and smooth ER. It is possible that the release of small VLDL follows the addition of a relatively small quantity of triglyceride (or of cholesteryl ester) to the nascent particle while large VLDL is formed by the addition of a substantial triglyceride core in a second, quantum step. Enhanced VLDL₂ secretion in moderate hypercholesterolemics may result from factors other than triglyceride availability, for example, the rate of cholesterol synthesis,¹³ cholesteryl ester availability,^{11 13 27} or microsomal transfer protein activity.²⁸

View larger version (26K): [in this window] [in a new window]   Figure 1. ApoB metabolism. ApoB is made constitutively in the liver. Initiation of lipoprotein assembly requires the addition of a small amount of neutral lipid to the growing polypeptide chain. Further lipid is added during particle assembly and secretion and the nature of the final lipoprotein released (which ranges from LDL to VLDL1) depends on the extent of lipidation. ApoB containing lipoproteins on entering the circulation are subject to lipolysis and remodeling so that the composition of the coat of the particle, especially its apoproteins, and the core change during the course of intravascular metabolism. Production rates for VLDL1 (Sf 60 to 400), VLDL2 (Sf 20 to 60) and IDL plus LDL-apoB are quoted in mg/d. Subjects with elevated plasma triglycerides (HTG) produce an excess of VLDL1 apoB (unpublished, 1997), whereas those with raised plasma cholesterol levels overproduce VLDL2 apoB.21 IDL plus LDL apoB production is variable over the range shown and is inversely related to the plasma triglyceride concentration.21

Cross-sectional surveys revealed that variation in plasma triglyceride is principally a function of changing VLDL₁ levels.²⁹ Likewise, it is larger VLDL that accumulate in hypertriglyceridemics and are lowered by fibrate therapy.^{30 31 32} The plasma concentration of VLDL₁ has been found to be controlled by both the rate of production and the rate of delipidation of the lipoprotein [References 30 and 31^{30 31} ; C.J.P. et al, 1997, unpublished data]. The former appears to be under the influence of hormones^{24 25} while the latter has a number of possible determinants. For example, investigation of the VLDL₁ to VLDL₂ conversion in subjects with genetic dyslipidemia revealed that the step was critically dependent on LpL, (the fractional transfer rate from VLDL₁ to VLDL₂ was reduced 90% in LpL deficiency^{22 33} ) but was not affected by HL deficiency,³⁴ by homozygous FH³⁵ or by homozygosity for apolipoprotein E₂.²⁰ From studies of VLDL composition,³⁶ human mutants,³⁷ and genetically altered mice,³⁸ it appears that apo-CIII as well as apo-CII is a modulator of LpL activity³⁹ (Fig 1). Recent kinetic experiments showed that VLDL₁ lipolysis but not that of VLDL₂ was inhibited profoundly by the presence of chylomicron-like emulsion particles in the circulation.⁴⁰ The implication of this finding is that VLDL₁ lipolysis does not "compete" effectively with chylomicron clearance but is virtually suspended when the plasma content of chylomicrons is high and then resumes when lipid absorption is complete. Presumably VLDL₁ is a much poorer substrate for LpL than dietary particles. It is not yet clear what regulates lipolysis in vivo, LpL activity as measured in post-heparin plasma showed only a weak correlation with either the extent of alimentary lipemia or VLDL₁ concentration.^{29 41} More likely it is the surface composition of VLDL, both its lipid and apoprotein content (eg, apoC-II/C-III or apoE/C ratios) that is critically important in most subjects.^{36 37 38 39}

VLDL₁ has two distinct metabolic fates, conversion by LpL to VLDL₂ and direct catabolism. The nature of the second process is unknown at present but it is quantitatively significant since up to half the apoB in a VLDL₁ tracer was removed directly from the circulation without appearing in denser fractions^{20 21 22} (Fig 1). Since the rate of VLDL₁ direct catabolism was similar to normal in FH homozygotes,³⁵ it is unlikely that the LDL receptor is involved. However, catabolism of the lipoprotein was reduced substantially in normolipemic apoE₂ homozygotes²⁰ and in subjects lacking LpL.³³ These proteins have been implicated in the binding of lipoproteins to agents such as the LRP and the VLDL receptor^{42 43} and the observations raise the possibility that these entities mediate the direct removal of large triglyceride-rich VLDL (Fig 1). The ligand for the putative receptor is likely to be apoE since apoB appears not to be in a receptor competent conformation on large triglyceride-rich VLDL.⁴⁴ By extrapolation from animal studies it is also probable that apoCIII plays an inhibitory role by interacting with or displacing apoE.^{38 45 46} The metabolic properties of VLDL₁ suggest that in many ways it acts as a liver-derived "chylomicron particle"; it is produced in response to the presense of triglyceride in the cell; and it is cleared rapidly by the same agents (initially LpL and then receptors) that metabolize dietary particles. The finding that VLDL₁ release is suppressed by insulin,²⁵ a hormone elevated during the absorption of fat in the diet, indicates that chylomicron and VLDL₁ secretion may be controlled in a reciprocal fashion. In this context it is noteworthy that some mammalian species release B-48 containing large VLDL when faced with the need to secrete increased quantities of triglyceride from the liver.⁴⁷

The VLDL₂ density interval (S_f 20 to 60) contains the products of VLDL₁ delipidation and newly secreted VLDL particles. The need to postulate synthesis of small VLDL by the liver was seen in kinetic studies where the amount of apoB generated by VLDL₁ delipidation was insufficient to account for the total mass seen in the density interval²³ and it was only by permitting VLDL₂ direct production that the kinetics of this lipoprotein fraction could be explained. As shown in Fig 1, ¹³¹I-VLDL₂ derived from injected ¹³¹I-VLDL₁ was found to be converted to IDL and LDL at a slower rate than a tracer of radio-labeled whole VLDL_2. This can only occur if a new lipoprotein species is introduced into the VLDL₂ interval. Furthermore, in a continuous separation medium such as a density gradient, a distinct peak was seen in the VLDL₂ range that strongly suggested the insertion of newly secreted lipoprotein.¹⁷ The possibility of metabolic channeling within the VLDL-LDL delipidation cascade was first mooted by Fisher,⁴⁸ and its confirmed presence^{23 49} indicates that the properties of a circulating lipoprotein depend heavily on its pedigree and that exchange of key lipid and protein components between particles is relatively slow compared to the processes of lipolysis and catabolism. VLDL₂ delipidation proceeded efficiently in subjects with LpL deficiency, evidence that the enzyme was not essential for the processing of this lipoprotein.³³ Likewise, in HL deficiency VLDL₂ was converted to IDL at about half the normal rate.³⁴ It is likely that both lipases contribute to this step (Fig 1). LDL receptors are likely to be responsible for direct catabolism of VLDL₂; smaller VLDL are more effective ligands than their larger counterparts for this receptor⁴⁹ and in vitro studies support the view that binding occurs via the apoB rather than apoE.⁵⁰ In line with this suggestion we found that VLDL₂ direct catabolism was similar to normal in apoE₂ homozygotes.²⁰ Further, while cyclohexanedione modification of apoB in S_f 60 to 400 VLDL that abolishes receptor mediated clearance did not affect its clearance rate (presumably because apoE mediates the receptor-lipoprotein interaction in this density interval⁴³ ), treatment of S_f 12 to 60 lipoproteins with this agent substantially retarded their removal from the circulation.⁵¹

	Properties of VLDL Subfractions Separated by Affinity Chromatography

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
Cell surfaces are coated with heparan sulfate and other similar compounds believed to facilitate the proximation of lipoproteins containing apoB and apoE to lipases and receptors. These apoproteins have specific glycosaminoglycan binding sites and so adhere to columns containing Sepharose-bound heparin. The affinity of VLDL fractions for heparin, however, is variable and dependent on the apoE content of particles and the conformation of apoB.^{52 53 54} In the case of the latter protein, expression of heparin binding domains, like the receptor binding site,⁵⁰ appears to be influenced by the size of the lipoprotein and its lipid composition. VLDL when applied to heparin-Sepharose is separated into bound and unbound fractions. Retained particles accounted for over half the VLDL mass in normolipemics, had a higher apoE: apoB ratio, were relatively rich in cholesteryl esters, and were smaller than unretained particles.^{52 53 54 55} The two fractions are present in variable proportions throughout the S_f 20 to 400 density interval, although VLDL₂ has been shown to have a substantially higher content of retained particles.⁵⁶ Early studies⁵⁷ of unretained VLDL showed that it was particularly rich in phosphotidylethanolamine(PE), a major phospholipid found in abundance in cell membranes but not in the majority of plasma lipoproteins and that it was a poor ligand for the LDL receptor, possibly because it was lacking in apoE but also due to the conformation of its apoB being inappropriate for binding. Fielding and Fielding on the basis of these properties suggested that unretained particles were newly secreted.⁵⁷ Co-incubation of apoE poor (ie, unretained) VLDL with retained apoE rich VLDL did not lead to significant inter-particle transfer of PE or apoE, and the same workers in a later investigation⁵⁸ reported that unretained VLDL required to be lipolysed to be able to bind apoE to its surface. This modification of VLDL was associated with, and was possibly dependent on, a perturbation in the structure of apoB. It was postulated⁵⁸ that unretained VLDL was the metabolic precursor to retained VLDL and that lipolysis was necessary to change apoB conformation and so reveal heparin binding sites and permit apoE to adhere to the particle (Fig 2). The hypothesis is consistent with data showing that in hypertriglyceridemic subjects, who are known generally to overproduce VLDL (Fig 1),⁵⁹ unretained VLDL accounted for 40% of total VLDL compared to 25% in normals.^{55 56} Treatment with diet or a fibrate led to a decrease in the proportion of unretained VLDL.⁵⁵ The requirement of a nascent particle to undergo lipolysis before it can, through a perturbation in the apoB conformation or through the acquisition of apoE, bind to receptors may be a mechanism by which futile cycling of VLDL is prevented in the liver. This organ secretes VLDL that could in theory be immediately removed by cell surface receptors especially since apoE is present in abundance on the surface of hepatocytes.⁶⁰

View larger version (16K): [in this window] [in a new window]   Figure 2. Metabolic scheme for VLDL subfractions isolated by heparin-Sepharose chromatography. VLDL can be fractionated by affinity chromatography on solid phase heparin into bound and unbound fractions. The former are retained either by virtue of the apoE on the particle or by the expression of a heparin binding site on apoB. Unbound VLDL show characteristics of nascent particles and when injected in vivo are converted to a bound species. In vitro evidence indicates that unbound VLDL must undergo lipolysis before apoE will adhere to the particle.58 The proportion of unbound VLDL is higher in the VLDL1 than in the VLDL2 flotation range.56

Metabolic studies reveal that when unretained VLDL was radiolabeled and injected into normal or hypertriglyceridemic subjects, it was converted into a VLDL that was able to bind to heparin and thereafter was delipidated to IDL.⁵² However, it was found that retained VLDL was not simply the product of lipolysis of unretained VLDL. Barrett et al⁵⁶ provided evidence for the direct input of both triglyceride and apoB into the bound and unbound fractions. Furthermore, their results were consistent with direct catabolism of unbound VLDL as well as lipolytic conversion to bound lipoproteins. Turnover experiments in rabbits and mini-pigs revealed that apoE-rich VLDL was catabolized at a greater rate than apoE-poor VLDL, principally by direct clearance from the circulation,^{53 56} and apoE-rich VLDL were less likely to be converted to LDL in rabbits than apoE-poor particles.⁶¹ These findings were consistent with the in vitro observations regarding the relative abilities of these lipoprotein fractions to interact with the LDL receptor.⁵⁴

Aware that VLDL particles may bind to heparin through both apoB and apoE, Campos et al⁵⁴ have recently examined the distribution of VLDL subfractions separated first on anti-apoE affinity columns and then on heparin-Sepharose. VLDL from subjects with a range of plasma triglyceride concentrations were first passed through the anti-apoE column; about 30 to 50% were unretained and hence were deficient in apoE. Chromatography of these "B" particles on heparin-Sepharose generated both bound (1% to 35% of total VLDL) and unbound (14% to 36% of total VLDL) fractions. The latter form of "B" particle was larger and more triglyceride-rich than either the bound "B" particles or the B/E particles whose lipid composition resembled each other. This was clear evidence that in earlier studies the retained heparin-Sepharose fraction was a mixture of "B"–and "B/E"–VLDL with the former binding by virtue of the apoB conformation (Fig 2). The apoC:apoB ratio was similar in all fractions isolated. Heparin-Sepharose unbound "B" particles failed completely to bind to the LDL receptor in vitro, while heparin bound "B" VLDL interacted with the receptor at an affinity approaching that of B/E VLDL. Indeed the affinity of VLDL for the LDL receptor was not related to the particles' apoB/apoE ratio. Campos et al⁶² have also shown that B/E VLDL can be subdivided by affinity chromatography on an antibody column that recognizes an epitope near the mid-portion of apoB polypeptide. This antibody appears to be selective for remnant particles and consideration must now be given to the possibility that the "signal" that a VLDL particle has become a remnant may reside in the conformation of apoB. Thus, there is now evidence that a minimum of four VLDL subfractions can be isolated on the basis of their apoE content and apoB conformation, so ample heterogeneity exists within VLDL to support the phenomenon of metabolic channeling described above.

It is difficult to relate unambiguously the structural and metabolic heterogeneity in VLDL revealed by centrifugation based fractionation to that based on affinity chromatography. VLDL₁ (S_f 60 to 400) has been shown to have a high content of apoE-poor particles that do not bind to heparin-Sepharose. This is consistent with the high triglyceride content and other attributes expected of new secreted VLDL. Heparin-Sepharose bound VLDL₁ may be remnants already formed within the density interval. Lipolysis of "B" particles to VLDL₂ leads to a change in the conformation of apoB and the acquisition of some apoE, thus rendering the VLDL able to bind to heparin. Thus, within the VLDL₂ density interval the relatively slowly metabolized remnants of VLDL₁ delipidation (Figs 1, 2) are likely to be heparin-Sepharose bound "B" or "B/E" particles. The nature of newly secreted VLDL within the S_f 20 to 60 is less clear. It is conceivable that heparin-Sepharose unbound VLDL₂ comprises, as in VLDL₁, nascent particles. These are reported to represent 10% to 50% of the VLDL₂ apoB mass,⁵⁶ a figure in line with the amount of the lipoprotein estimated from kinetic studies to be present in newly secreted material.^{20 21} Hence, it is tempting to speculate that heparin unbound VLDL of both density intervals is the form in which particles are released from the liver (Fig 2). However, Barrett et al⁵⁶ required newly synthesized lipoprotein to enter both heparin-bound and heparin-unbound VLDL₂ to fit the model to the observed data and so the form of nascent particle remains an open question. The metabolic fate of heparin-bound versus heparin-unbound VLDL has not been followed through to LDL in man, and so it is not yet obvious which particles act as precursors to the latter lipoprotein. Analogy with rabbit studies suggests that those particles that acquire sufficient apoE will be efficiently removed by receptors and this presumably occurs throughout the S_f 0 to 400 range. Others will progress down the delipidation cascade to form LDL.

	Heterogeneity of Intermediate Density Lipoproteins

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
Kinetic studies in the mid-1970s identified IDL as a class of lipoprotein with distinct metabolic properties that acted as a transient intermediate in the delipidation cascade from VLDL to LDL. Subsequently it was shown that not all IDL were derived from VLDL and not all IDL were converted to LDL, a portion of the lipoprotein was catabolized directly from plasma probably by the LDL receptor since the rate of this process was dramatically reduced in FH homozygotes.^{35 63} Turnovers conducted in subjects with genetic dyslipidemia indicate that hepatic lipase was essential for the IDL to LDL conversion.³⁴ Similarly, infusion of antibodies to HL in animals gave rise to an accumulation of S_f 12 to 20 lipoprotein and a decrease in LDL.⁶⁴ The IDL to LDL conversion was also found to be dependent on the apoE phenotype of a subject. In a study of normolipidemic apoE₂ homozygotes we observed a 60% reduction in the rate of transfer of IDL to LDL while direct catabolism of the fraction, presumably mediated by its apoB component, was normal.²⁰ The diminished circulating LDL mass in apoE₂ homozygotes was the result of this failure to delipidate IDL efficiently. Unexpectedly the IDL to LDL lipolytic step was also greatly impaired in FH homozygotes. Up to 5 days was required to complete the delipidation of VLDL to LDL in such subjects³⁵ compared to less than 1 day in normals. Thus, the mechanism of conversion appeared to be independent of LpL³³ but involved the possibly concerted action of HL, apoE_3, and the LDL receptor. It is conceivable that on the liver sinusoidal cells when HL is sited, an interaction between apoE and the LDL receptor facilitates the delipidation step. About half of the apoB entering the IDL density interval was transmitted to LDL in normals and half is removed by catabolism.²⁰ What determines the metabolic fate of the particles is unknown. It could be due to structural heterogeneity (the basis of which is as yet unrecognized) or to the relative likelihood of IDL particle binding to HL and being lipolysed to LDL or binding to the LDL receptor located on a hepatic parenchymal cell and being internalized and degraded.

Krauss and co-workers in a series of investigations^{65 66} have examined the structural and metabolic heterogeneity of IDL. They reported the presence on gradient gel electrophoresis of two major subfractions that overlapped in size and density and hence cannot be readily isolated. IDL-1 were larger (280 Å to 300 Å in diameter), less dense and seemed to form a component of a spectrum of particles that ranged from S_f 14 to 60, ie, into the VLDL₂ density range. This species was relatively triglyceride-rich whereas IDL-2 was smaller (270 Å to 280 Å in diameter) and contained relatively more cholesterol. Given the fact that both VLDL and LDL exhibit a high degree of heterogeneity it is entirely likely that the IDL class also contains discrete species with individual metabolic properties. These have been examined in a rat model system by Musliner et al.⁶⁶ IDL-1 appeared to give rise to intermediate-sized LDL particles while conversely IDL-2 was the precursor to the largest LDL species, LDL-1. These nonintersecting pathways of IDL and LDL interconversion have been formulated into a metabolic scheme by Krauss.⁶⁷ The importance of IDL lies not only in the fact that it is the immediate precursor to LDL but that it appears to be particularly atherogenic. A number of studies have linked raised IDL levels to increased risk of CHD as recently summarized by Superko.⁶⁸

	LDL Structural and Metabolic Heterogeneity

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
LDL as the major cholesterol-carrying lipoprotein in plasma is the fraction most strongly implicated in atherogenesis. When examined in the analytical ultracentrifuge "shoulders" were apparent on the LDL peak suggestive of the presence of subfractions of differing flotation rate and early reports from Fisher⁴ highlighted the polydispersity of LDL in hypertriglyceridemia compared to the "monodisperse" distribution seen in normals or subjects with elevated cholesterol levels. It was considered for many years that LDL comprised a population of particles with continuously variable size and density across the range of 200 Å to 270 Å and d 1.019 g/ml to 1.063 g/ml. However, Krauss⁶⁹ using the high resolution technique of gge provided convincing evidence that in virtually all subjects LDL was made up of a small number of subtypes of particles with relatively discrete size and density, ie, it was "paucidisperse." A seminal observation was that patients with a preponderance of small-sized LDL detected by gge (pattern "B" LDL) had a three-fold increased risk of having a myocardial infarction independent of the total concentration of LDL present.⁷⁰ This finding triggered the current widespread interest in LDL size as a predictor of CHD risk and the association has been confirmed many times.^{71 72 73} Since small, dense LDL is clinically important it is essential to understand its origins and the basis for its increased atherogenic potential compared to larger species of the lipoprotein. At present opinions are divided as to whether a high concentration of small, dense LDL in the plasma is the result of a specific inherited trait⁶⁸ or if the particle subtype is generated in anyone given the appropriate conditions. In the discussion that follows, evidence is presented to support mainly the latter view, although in some individuals it is conceivable that an overriding genetic factor may be present to cause the generation of small LDL.

	LDL Particle Size and Plasma Triglyceride

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
From the earliest studies of LDL heterogeneity, whether assessed by size on gradient gels or by density in a centrifuge rotor, it was noted that subjects with smaller and denser LDL had higher plasma triglyceride levels than those with lighter particles. Austin et al in a classic paper⁷⁰ found that pattern B was associated with a two-fold increase in plasma triglyceride, higher plasma apoB and IDL levels and reduced HDL cholesterol and apoA-I concentration, ie, small, dense LDL did not appear in isolation from other plasma lipid abnormalities. Campos et al⁷³ reported a highly significant correlation between LDL size score (a continuous index of LDL diameter rather than the dichotomous classification of Austin and Krauss⁷⁰ ) and plasma triglyceride. Further, they showed that once triglyceride was taken into account, LDL size was not an independent discriminator of CHD risk. Methods based on centrifugation of LDL in a density gradient were developed by Swinkels et al,⁷¹ Chapman et al,⁷⁴ and ourselves⁷² in an attempt to quantify the amount of various LDL subfractions present. Although some workers divided LDL into multiple fractions,⁷⁴ it was clear that if plasma (rather than prepared LDL) was applied to a gradient and the subfractions generated rapidly that three discrete fractions were present in most normal or moderately hyperlipidemic subjects [LDL-I, d=1.025 g/ml to 1.034 g/ml, LDL-II, d=1.034 g/ml to 1.044 g/ml and LDL-III, d=1.044 g/ml to 1.060 g/ml (nb, these ranges vary slightly between publications)] while in severe hypertriglyceridemics very small and dense species were observed (LDL-IV⁶⁹ ). The link between plasma triglyceride and LDL size phenotype was first explored by Austin et al.⁷ They found that pattern A (large LDL predominant) was universally present at low plasma triglyceride levels (<0.5 mmol/l) whereas pattern B was found in most individuals whose triglyceride exceeded 2.0 mmol/l. Similarly, McNamara et al⁷⁵ showed that change in LDL size was related to change in plasma triglyceride level. However, it is necessary to turn to a continuous, quantitative assessment of LDL subfraction concentrations in order to uncover the precise nature of the relationship between plasma triglyceride and LDL size. In our laboratory LDL subfraction concentrations were measured in a large number of normal and CHD affected subjects.^{29 72} In the study of Griffin et al,⁷² it was found that LDL-I and LDL-II had similar plasma concentrations in those with and without CHD but LDL-III was 2-fold higher in affected subjects. An LDL-III level of 100 mg/dl was the best discriminant between the two groups and was associated with a 7-fold increase in risk.⁷² Close examination of the relationships between LDL subfraction concentrations and plasma triglyceride revealed that LDL-I decreased as plasma triglyceride rose across the normal range. LDL-II showed a biphasic association with a positive correlation below a value of 1.5 mmol/l for the plasma lipid and a negative one above that (Fig 3A). LDL-III remained below 100 mg/dl until the plasma triglyceride exceeded 1.5 mmol/l and then the level of the subfraction increased dramatically. In both surveys^{29 72} the LDL-III-plasma triglyceride curve gave the impression of a breakpoint at the level of about 1.5 mmol/l, at least in male subjects. This concurred with the earlier observation of Austin et al⁷ that pattern B only becomes prevalent above this value. The fact that the total LDL concentration remained relatively constant in subjects with plasma triglyceride in the range 1.0 mmol/l to 3.0 mmol/l suggested to us that 1.5 mmol/l was a "threshold" above which either LDL-II was increasingly converted to LDL-III in the circulation or LDL-III rather than LDL-II was the preferred product of VLDL delipidation. The postulated existence of a threshold concentration of plasma triglyceride rich lipoproteins for the formation of small, dense LDL was intriguing and helped explain the observation of Superko and Krauss⁷⁶ that nicotinic acid treatment of moderately hypercholesterolemic subjects led to a change in LDL size from pattern B to A only if the plasma triglyceride fell below 1.6 mmol/l. Similarly in a number of later drug studies, reduction in plasma triglyceride led to a diminution of LDL-III and an increase in LDL-II even if total LDL mass was unaltered on therapy [Reference 77⁷⁷ and M.J. Caslake et al, 1997, unpublished data (Fig 3B)]. Further evidence for a threshold effect in the conversion of LDL-II to LDL-III has been observed during gestation.⁷⁸ Plasma triglyceride climbed from 1.0 mmol/l to 2.5 mmol/l from the first to third trimester and the LDL concentration rose 70%. At first LDL-II accumulated but as plasma triglyceride exceeded about 1.8 mmol/l in the group of women studied, there was an abrupt change in the relative concentration of LDL-II and LDL-III and at term most subjects exhibited a high LDL-III level.⁷⁸ The appearance of pattern B at the end of pregnancy and the reversion of LDL to pattern A postpartum has been reported by Silliman et al.⁷⁹ These investigations reveal the malleability of LDL subfraction profile and also raise the question as to the mechanism that underlies a step change in LDL size.

View larger version (18K): [in this window] [in a new window]   Figure 3. Influence of plasma triglyceride on the concentration of intermediate sized (LDL-II) and small dense LDL (LDL-III) in normal and hyperlipidemic subjects.

In our and others' experience⁸⁰ few individuals display small, dense LDL at low normal plasma triglyceride. The vast majority of the population who express pattern B-LDL or a LDL-III >100 mg/dl do so against a background of disturbances in lipoprotein metabolism linked to high normal or moderately elevated plasma triglyceride levels with attendant changes in IDL and HDL. The term atherogenic lipoprotein phenotype(ALP) was coined by Austin et al⁷ to describe a syndrome of small, dense LDL, moderately elevated VLDL and low HDL. It is increasingly recognized as a significant, and possibly in population terms the most important, lipid-associated risk factor for CHD. As described in following sections, elevation in plasma triglyceride due to increased VLDL₁ levels may be the key metabolic disturbance that gives rise to the syndrome.

	LDL Particle Size and Lipases

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
Generation of small, dense LDL requires the removal of lipid from the particle while, of course, apoB is retained. LDL is cholesteryl ester rich and since plasma is deficient in esterases that hydrolyze this lipid species, it is predictable that particles in which the core is predominantly cholesteryl ester are resistant to shrinkage. However, the action of CETP mediates the exchange of neutral lipid between lipoproteins with the result that when circulating levels of triglyceride-rich lipoprotein (VLDL, chylomicrons) are high, a portion of their core triglyceride is transferred into LDL (and HDL) and replaced with cholesteryl ester from the denser species (Fig 4). Triglyceride-enriched LDL is then a substrate for endothelial-bound lipases and their action leads to the formation of smaller, lipid-depleted particles. Cycles of neutral lipid transfer and lipolysis have been shown to promote the formation of HDL₃ from HDL₂⁸¹ and there is reason to believe that the same phenomenon occurs in the LDL density range.^{67 82} Either lipoprotein lipase or hepatic lipase(HL) could, in theory, hydrolyze triglyceride in LDL. In vitro and in vivo evidence strongly suggests that the latter enzyme is the more likely agent. It has a much higher affinity for LDL than lipoprotein lipase and has the capability to act as a phospholipase as well, hence removing both core and surface components from the particle.^{83 84}

View larger version (20K): [in this window] [in a new window]   Figure 4. Model integrating LDL structural and metabolic heterogeneity. Kinetic evidence suggests that the two metabolically distinct pools in LDL ( and ß) arise from different sources. Pool is the major species detected by multicompartmental modelling of subjects with low normal plasma triglyceride levels. It is postulated to arise when apoB is secreted into the Sf 0 to 60 density range. LDL with the kinetic properties of pool ß has been shown to be the product of VLDL1 delipidation.20 21 The two LDL species have substantially differing residence times (RT) in the circulation. LDL-III generation is favored when plasma triglyceride exceeds 1.5 mmol/l and HL is in the male range (i.e. >15 u/l29 ). VLDL1 is the principal species that accumulates as plasma triglyceride levels rise in the population29 as a result of overproduction of the lipoprotein or its defective removal (LpL or elevated apoCIII levels). It is envisaged that above 1.5 mmol/l threshold enough VLDL1 is present to both produce long-lived pool ß LDL and to cause triglyceride enrichment of the particle to a level that makes LDL-III formation possible. The action of HL removes lipid from the LDL-II to form LDL-III.

The possibility that LpL was involved in the regulation of LDL size was investigated by Karpe et al,⁸⁵ who found a positive association between LpL activity and the amount of LDL in a light (d <1.040 g/ml) fraction. Furthermore, obligate heterozygotes for LpL deficiency show multiple lipoprotein abnormalities including pattern B LDL.⁸⁶ However, these associations are likely to have been mediated through the effect of LpL variation on chylomicron and VLDL concentrations (Fig 4). Hepatic lipase activity, on the other hand, was shown to be positively related to the amount of small, dense LDL⁸⁷ although this association was not confirmed in the subsequent study of Jansen et al.⁸⁸ We explored the relationship between lipases and LDL subfraction concentration in normolipemic men and women and in an initial study⁸⁹ were able to show a strong correlation between HL activity and the plasma concentration of LDL-III. Moreover, there was a strong suggestion that common factors regulated the subfraction distribution in LDL and HDL. In a larger number of subjects,²⁹ it was observed that women at plasma triglyceride levels above the indicated threshold for the formation of small, dense LDL (ie, about 1.5 mmol/l) had less LDL-III than men (Fig 3A). Multivariate analysis showed that in men the LDL-III concentration was explained mainly by plasma triglyceride level but in women HL activity and plasma triglyceride were both determinants. Apparently for LDL-III to rise above 100mg/dl in females it was necessary for HL to be in the male range. Normally the HL level in women is half that seen in men, a result that is readily explained by the regulatory influence of sex hormones on the enzyme.²⁹ The discrepant results in the study of Jansen et al⁸⁸ can be explained by the fact that they examined only men. Further support for a key role of HL was seen in the condition of inherited deficiency of the enzyme; affected subjects had large, buoyant LDL subfractions in plasma.⁹⁰ These findings led us to postulate a model in which the formation of a significant amount of LDL-III in plasma (ie, above 100 mg/dl) required the combination of a plasma triglyceride >1.5 mmol/l and a HL >15 u/l (Fig 4). The model helped to explain the reduced penetrance of pattern B LDL in females (low HL activity) and in younger subjects^{7 91} (plasma triglyceride climbs dramatically in the population from about 0.5 mmol to 1.0 mmol at age 20 to 1.5 mmol/l to 2.0 mmol/l at age 40).⁹² Alteration in HL activity may alter the "threshold" seen in the plasma triglyceride - LDL-III association. Thus, a high HL in a man may result in the generation of small, dense LDL at plasma triglyceride less than 1.5 mmol/l. Conversely, an elevated plasma triglyceride may cause LDL-III to form even in women with normal HL levels, eg, as in pregnancy. If the model is correct, then the mechanistic implication is that the formation of small-sized LDL depends on the rate of transfer of triglyceride molecules into LDL (a function principally of the concentration of triglyceride-rich lipoproteins in plasma since CETP activity is not normally rate-limiting⁹³ ) and the rate of their hydrolysis by HL. The susceptibility of LDL to cycles of triglyceride enrichment and hydrolysis has been demonstrated in vitro by Lagrost et al.⁸² The suggested existence of a threshold indicates that these rates must achieve certain values before significant quantities of normal-sized LDL are converted to their smaller counterparts. Also, the symmetry of the mechanisms for formation of small, dense species in LDL and HDL^{81 89 94} helps to explain why low HDL levels are a component of an ALP and why HDL cholesterol is strongly inversely correlated with LDL size.^{29 73 75 80}

	LDL Heterogeneity and CETP

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
CETP deficiency is an extremely rare inherited condition mainly found so far in the Japanese population. It is associated with high levels of HDL cholesterol (3 mmol/l to 4 mmol/l) but relatively normal levels of other plasma lipoproteins.⁹⁵ Investigation of LDL structure in affected subjects revealed the presence of two major species, one of near normal size and the other much smaller than normal.⁹⁶ From the gradient gel electrophoresis it appeared that both co-existed throughout almost the entire LDL density range and IDL also showed two components on gradient gel electrophoresis. LDL were found to be cholesteryl ester poor and triglyceride rich and Sakai et al⁹⁶ proposed from these observations that VLDL was secreted by the liver as two discrete species, which were delipidated through nonintersecting pathways through IDL to LDL. The metabolic model described in Fig 1 accords with this suggestion. They further postulated that the small LDL in their patients normally received cholesteryl ester from HDL via CETP and so maintained (or attained) a size comparable to that of the larger LDL species (Fig 4). CETP may, therefore, have a dual action on the LDL subfraction distribution. That is, it facilitates the transfer of cholesteryl esters from HDL to LDL and this may be the major fate of this lipid in subjects with low levels of triglyceride-rich lipoproteins (the other potential acceptor for cholesteryl esters) and in the fasting state. This action favors the formation of larger LDL and as noted above the maintenance of such species in the circulation. If, however, plasma triglyceride levels rise CETP mediates other transfers that lead to a reduction in LDL size (Fig 4). The intriguing observation that not all LDL is affected uniformly by CETP deficiency may be explained by the presence of metabolically distinct species in this density range.

Excess alcohol intake leads to a secondary, partial CETP deficiency and has been shown to be associated with the appearance of multiple LDL species on gge. Heavy drinkers with <25% of normal CETP activity had small-sized as well as normal-sized LDL and on abstention from alcohol the pattern normalized.⁹⁷ The authors of this report also noted that in obligate heterozygotes for CETP deficiency where CETP levels were half normal the LDL pattern was not disturbed, thus a marked decrease in activity was required before very small LDL species were seen. It is of interest to note that the only other patient group where LDL are so small is severe hypertriglyceridemics^{69 98} and it is tempting to speculate that in this situation also there is a markedly reduced transfer of HDL cholesteryl ester into LDL since VLDL and chylomicrons are present in large quantities to act as preferential acceptors. The role of CETP as a determinant of LDL size has been explored in detail by Guerin et al⁹⁹ and Lagrost et al.¹⁰⁰ Guerin et al¹⁰¹ identified in experiments in vitro that it was larger triglyceride-rich LDL subfractions that were the preferential acceptors of CETP-mediated transfer of HDL cholesteryl ester in plasma from normal subjects. Absence of transfer activity in CETP deficiency presumably led to this LDL species in particular becoming delipidated by the action of HL to small sized particles.

	Genetic Influences on LDL Heterogeneity

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
LDL size pattern B appears to be an inherited trait. Austin and Krauss who have conducted many studies on this topic^{7 91 102 103 104} showed that about 30% of men exhibit this pattern and that in families its appearance was due to a major gene effect with a dominant or additive mode of inheritance. There was reduced penetrance in men under 20 years old and women under 50 years,^{80 91} possibly explained by the model postulated above. Examination of families with familial combined hyperlipidemia revealed that transmission of pattern B (with an allele frequency of 0.25) was independent of plasma apoB levels but closely linked to the presence of elevated plasma triglyceride.¹⁰² Twin studies indicated that about a third to a half of the variation seen in the LDL subfraction profile was due to genetic factors with the remainder being attributed to hormonal, nutritional, and environmental influences.^{80 104} From the proposed model for the generation of small, dense LDL it is obvious that there are a substantial number of candidate genes that could be responsible for inheritance of pattern B, eg, variation in LpL or apoCIII may elevate plasma triglyceride above the threshold whereas variation in HL or CETP may have a more direct effect on LDL structure. Linkage studies have associated pattern B with polymorphisms in or near the LDL receptor, Apo AI-CIII-AIV, CETP, and manganese superoxide dismutase loci[reviewed in 80]. A relationship to the common apoE polymorphism has, at least in one study, been ruled out.¹⁰⁵

Numerous investigators have reported that pattern B on gge or a high concentration of LDL-III is very common in subjects with insulin resistance or frank noninsulin dependent diabetes mellitus.^{106 107 108} Likewise, individuals with pattern B LDL have the hallmarks of insulin resistance compared to those with pattern A.¹⁰⁹ The link between insulin resistance and phenotype B is so strong that the latter has been added to the list of abnormalities that characterize the "insulin resistance" or "metabolic" syndrome.¹⁰⁹ However, it is likely that the connection between the two inherited traits is through plasma triglyceride elevation. In a study where NIDDM patients were divided into those with (mean plasma triglyceride 2.91 mmol/l) and without (mean plasma triglyceride 1.48 mmol/l) raised VLDL levels, the former but not the latter had a high LDL-III concentration despite the fact that both groups had similar HbA₁, glucose and insulin concentrations and an equal degree of insulin resistance.¹⁰⁸

	Metabolic Heterogeneity of LDL

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
Berman, the pioneer of multicompartmental modeling of lipoprotein metabolism, was the first to point out that LDL apoB kinetics did not fit the classical paradigm for a plasma protein.¹¹⁰ The key observation was that although the plasma decay curve of a radioiodinated LDL tracer was biphasic as for other proteins, the rate of urinary excretion of degradation products (iodiotyrosine) was nonuniform, higher than predicted at the beginning of the turnover and lower at the end. The ratio of the amount of radioactivity excreted in urine in 24 hours divided by the mean plasma radioactivity in the same period (U/P) is a daily measure of the metabolic potential of the protein under investigation. For a metabolically homogeneous substance like albumin the FCR calculated from the plasma decay curve is equal to the daily U/P ratio, which is constant. However, it was observed that after injection of an LDL tracer the U/P ratio in a typical normolipemic subject fell from about 0.5 at day 2 of the turnover to about 0.25 at day 12 (Fig 5B).¹¹¹ This indicated that LDL was metabolically heterogeneous with one component being removed rapidly during the early phase of the experiment, ie, the initial rapid plasma decay is due to both intra-extravascular exchange of LDL and catabolism of the lipoprotein. Foster et al¹¹² developed a number of models to account for this phenomenon in hypertriglyceridemic subjects and we adopted one of these¹¹¹ to explain the metabolic behavior of LDL in normals and hyperlipidemics in the basal and treated states.^{77 111 113} We also found that tracer ¹³¹ I-cyclohexanedione treated LDL that does not bind to receptors gave a relatively constant low daily U/P ratio¹¹¹ (Fig 5B), evidence that the early rapid clearance of LDL from plasma was due to the action of LDL receptors. Thus, we interpret the U/P and plasma decay curves for LDL as follows: at the beginning of a turnover the tracer contains two metabolic components, and ß (pool and ß are used here instead of A and B as in References 77, 111, and 113^{77 111 113} , to avoid confusion with the LDL size pattern nomenclature); pool is rapidly cleared by receptors, leaving with time an increasing proportion of pool ß, a species with a relatively low affinity for receptors. Others have developed similar models¹¹⁴ and there is general agreement about the meaning of these turnover data.

View larger version (32K): [in this window] [in a new window]   Figure 5. Relationship between VLDL and LDL apoB kinetic heterogeneity. A, LDL cholesterol levels (, dotted line) change dramatically as a function of plasma triglyceride (note log scale). This is due first to a fall-off in the FCR for LDL apoB as plasma triglyceride rises from 0.5 to 1.8 mmol/L and then an increase in the FCR above this value (, dashed line). The lines are quadratic fits to data taken from References 77, 112, and 113, and unpublished work. B, The ratio of urine radioactivity excreted compared with the mean plasma radioactivity (U/P) following injection of a tracer of radioiodinated LDL is a daily measure of the catabolic rate of the lipoprotein. If the protein under study is kinetically homogeneous, it should be a constant value over the whole period of the turnover. The fall-off in U/P ratio in a subject with a low normal () or an average () plasma triglyceride indicates metabolic heterogeneity, with the early loss of a rapidly removed species (pool ) and an increasing proportion of slowly cleared (pool ß) LDL remaining. The dashed line shows the U/P ratio for a cyclohexanedione-treated LDL tracer that does not bind to receptors.111 C, Appearance of radioactive apoB in LDL following simultaneous administration of VLDL1 and VLDL2 tracers.23 LDL generated by VLDL1 delipidation appears in and is cleared from LDL more slowly than LDL formed from VLDL2. A subfraction within VLDL2 is responsible for the rapid, efficient formation of an LDL species, which is itself removed relatively rapidly from the circulation (see Fig 1). D, Correlation between pool ß LDL production rate estimated from multicompartmental modeling of LDL turnovers and plasma triglyceride. () are values from normal and hyperlipidemic subjects in the basal state. Crosses are the values obtained when the hyperlipidemic patients were given fibrate treatment [References 77, 111, and 113, and unpublished data]

In a search for factors that influence LDL metabolic heterogeneity it was observed in a group of young subjects whose plasma lipid levels varied across the normal range that the nature of the U/P fall away curve changed with the triglyceride level.¹¹¹ At low levels of plasma triglyceride (<1.0 mmol/l), the decrease in daily U/P ratio from day 2 to 12 was steep whereas at higher triglyceride levels the initial U/P peak value was lower and the decline over the period of the turnover less marked (Fig 5B). Furthermore, in all subjects the U/P ratio at the end of the turnover was very similar, at about 0.2 pools/d to 0.25 pools/d. Multicompartmental modeling of the plasma and urine data allowed an estimation of the relative mass of apo LDL in pool and pool ß. The former changed little in the normolipemic group but pool ß mass rose markedly as plasma triglyceride rose.¹¹¹ Since the FCR for this pool was remarkably constant in normal and hyperlipidemic subjects^{77 111 113} then the level of pool ß was controlled by its level of production that showed a strong positive correlation with plasma triglyceride level (Fig 5D). It is a longstanding but not widely appreciated observation that the plasma LDL level exhibits a biphasic relationship to plasma triglyceride.^{111 115} As levels of triglyceride in the population rise from 0.5 mmol/l to 2.0 mmol/l, LDL cholesterol increases about 100% and then above a plasma triglyceride of 3.0 mmol/l, LDL decreases. This variation in LDL concentration is associated with a reciprocal change in the FCR for the lipoprotein (Fig 5A). Within the normal triglyceride range the fall in FCR is linked to a decreased rate of receptor-mediated catabolism.¹¹¹ In theory, reduction in the activity of receptors themselves could be invoked to explain this phenomenon but in light of the metabolic heterogeneity, described above, we favor an explanation in which the proportion of slowly metabolized pool ß increases relative to the rapidly cleared pool , ie, a change in ligand rather than receptor is responsible for the plasma triglyceride-FCR association. Further, the rise in the LDL concentration as plasma triglyceride rises across the normal range may be attributed to an increase in pool ß in the circulation (Fig 5A). In hypertriglyceridemics, the increase in LDL FCR (Fig 5A) was associated with a concomitant rise in the FCR for cyclohexanedione-treated LDL,¹¹⁶ while receptor-mediated clearance was constant over the range 2.5 mmol/l to 5.0 mmol/l of plasma triglyceride. These subjects have particularly small, dense LDL (LDL IV) that, it was suggested, are catabolized rapidly by receptor-independent pathways.¹¹⁶

The proportions of pool and ß can be perturbed in patients treated with plasma triglyceride-lowering drugs such as fibrates. Subjects with moderate hypercholesterolemia had a mean pool and ß masses of 1200 mg and 2100 mg as computed by modelling.¹¹³ Fenofibrate treatment led to a specific decrease in pool ß to 1200 mg with little change in pool (1100 mg on drug). The FCR of pool increased from 0.39 pools/d to 0.66 pools/d, whereas that of pool ß was unaltered at 0.2 pools/d. A similar effect was observed in a separate group treated with ciprofibrate.⁷⁷ Again, the plasma triglyceride both on and off therapy was strongly associated with the amount of pool ß production (Fig 5D). These findings indicate that the plasma triglyceride (ie, VLDL) concentration is the principal determinant of pool ß abundance in the LDL density range as well as the major factor regulating the LDL-III concentration. Thus, it is likely that structural and metabolic heterogeneity are intimately related. However, our first supposition that pool ß was located in LDL-III was considered unlikely since the mass of pool ß apo LDL exceeded in many subjects the estimated amount of apoB in LDL-III.

	Linking VLDL and LDL Metabolic Heterogeneity

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
VLDL and LDL apoB kinetics show clear evidence of metabolic heterogeneity as described above and it is likely that they are linked. In dual tracer studies of the VLDL to LDL conversion it was repeatedly observed that the LDL derived from delipidation of ¹³¹ I-VLDL₁ was cleared relatively slowly from the circulation. Indeed, in a group of 21 moderate hypercholesterolemic subjects we observed that the mean FCR for LDL apoB was about 0.2 pools/d and that this rate was uninfluenced by plasma triglyceride level.²¹ When ¹²⁵I-VLDL₂ was injected into the same subjects a portion of the tracer (as explained in section 2 and Reference 23²³ ) appeared rapidly and was cleared relatively quickly from the LDL density range (Fig 5C). As shown in Fig 1 this LDL is the product of a lipoprotein secreted first into the small VLDL (S_f 20 to 60) range. The FCR of LDL derived from VLDL₂ showed an inverse trend with plasma triglyceride level.²¹ From these findings we postulate that VLDL₁ is precursor to pool ß LDL. This link explains the direct association of plasma triglyceride with pool ß production (Fig 5D) since VLDL₁ is the major species that accumulates in the circulation as plasma triglyceride levels rise.²⁹ Furthermore, the agreement between the experimentally observed FCR for LDL derived from VLDL₁ and the mathematically derived FCR for pool ß LDL is evidence that the two plasma compartment model for LDL heterogeneity is correct.^{77 111 113}

The question remains as to how pool is generated. Kinetic studies over many years have shown that apoB containing lipoproteins are secreted not only in the VLDL density range but also in IDL and LDL.^{21 49 117} There has been controversy on the issue with some workers suggesting that what appears to be direct IDL/LDL synthesis is actually rapid delipidation of a small precursor pool of VLDL that is not labeled efficiently in the ex vivo iodination procedure.¹¹⁸ However, recent stable isotope experiments^{119 120} and earlier investigations that used radioactive amino acids⁴⁹ and hence were not subject to labeling artifacts have also provided evidence that apoB can be secreted throughout the entire S_f 0 to 400 range. In both radioactive²¹ and stable isotope tracer studies (C.J.P. et al, 1997, unpublished data) we have been able to show a strong inverse correlation (r = -0.55 and r = -0.60 in the two studies, both P < .02) between the percent of apoB secreted in the IDL/LDL density interval and plasma triglyceride concentration. These data taken together with other observations¹²¹ suggest, as first postulated in 1977,¹¹⁷ that a spectrum of apoB containing particles are secreted from the liver and that the nature of the particle is related to the plasma triglyceride, ie, at low levels of plasma triglyceride (<1.0 mmol/l) a substantial portion (up to 50%) of apoB is released as IDL or LDL²¹ but at high normal levels of the lipid more than 90% of apoB is secreted in the form of VLDL, principally VLDL₁^{20 21} (Fig 1). The further finding (C.J.P. et al, 1997, unpublished data) that in normal subjects with high IDL/LDL secretion rates the LDL FCR was also high (0.5 pools/d to 0.8 pools/d) indicated that this pathway is a source of pool LDL. Directly secreted VLDL₂ is also, according to the model in Fig 1, delipidated to LDL with a relatively rapid FCR and hence it is hypothesized that pool LDL is, in fact, generated following the release of lipoproteins in the S_f 0 to 60 density interval. Thus, kinetic heterogeneity in LDL is linked to the nature of the precursor lipoprotein as depicted in Figs 1 and 4.

	Linking Structural and Metabolic Heterogeneity in LDL

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
Implicit in the results of LDL turnovers that examine heterogeneity of the lipoprotein is that within the same density interval, even if "narrow-cut" LDL is used as tracer, there exist particles with differing metabolic fates.¹¹¹ There must be a structural explanation for this phenomenon and, with analogy to the situation for VLDL described in section 2, it is likely to be related to apoprotein content and/or conformation. One possibility is that pool LDL is enriched in apoE since this protein is able to enhance a particle's ability to bind to receptors. However, the reported content of apoE, especially of an intermediate-sized LDL (d 1.030 g/ml to 1.050 g/ml as used in most LDL turnover experiments) is very low, about 4 moles of apoE for every 100 particles¹²² and, therefore, cannot be the basis for the major division between pool and ß. ApoB is the primary ligand for receptors in small-sized VLDL⁵⁰ and LDL and there is now an abundance of evidence that its conformation is highly malleable and dependent on the particle's size and lipid content. It is, therefore, tempting to speculate that the basis of the pool , ß metabolic difference is a conformational variation in apoB so that in the former the receptor recognition site(s) are fully competent, whereas in the latter a critical domain is hidden. Chatterton et al¹²³ in an elegant study showed that apoB has a "ribbon and bow" structure on LDL and went on to suggest that the inability of VLDL apoB to bind to receptors was due to the "bow" portion covering the binding site around amino acid 3500. Lipolysis was postulated to alter this structural feature so that the critical domain was revealed. An addition to this hypothesis to explain the data presented above is that apoB released on S_f 0 to 60 particles has a different starting conformation from that released on S_f 60 to 400 lipoproteins and that a fully competent receptor site is formed when LDL are made from the former particles but not during the extensive delipidation necessary to turn the latter into LDL.

Plasma triglyceride is, therefore, the most important determinant of both the subfraction distribution of LDL and its metabolic heterogeneity. It seems reasonable to suggest that the metabolic conditions in subjects with high-normal or elevated plasma triglyceride are those that favor the formation of small, dense LDL. If, as shown by the studies described above, there exists within the LDL density range particles with a residence time of about 2 days (residence time is the reciprocal of the FCR) and others with a residence time of 5 days, there is more time for the latter to undergo cycles of neutral lipid exchange and HL action and these must be the favored substrate for LDL-III formation as depicted in the integrated metabolic and structural model shown in Fig 4. It resembles a scheme recently presented by Krauss⁸⁰ with the addition of quantitative conditions necessary for the generation of small, dense LDL.

	Properties of LDL Subfractions

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
There is a general tendency for LDL lipid content to reduce relative to protein as LDL particle size decreases,^{69 74 124 125} which is predictable on the basis of the pseudomicellar structure of lipoproteins and the density change. From lightest to densest particles the core composition changes from principally cholesterol ester to one that is more triglyceride-rich. McNamara et al¹²⁵ computed the predicted thickness of apoB on the surface of LDL and found it decreased from 24Å on larger to 16Å on smaller particles. This was thought to reflect a release of subdomains of apoB from interaction with the lipid core. The conformation of apoB in LDL has been repeatedly shown to be related to particle density even in normal subjects¹²⁶ but alterations in the structure of the protein are most evident in the small LDL found in hypertriglyceridemics where changes occur in the circular dichroism spectrum, epitope expression, and susceptibility to limited protease attack.^{127 128} Some workers have linked the perturbations in apoB structure to the particle's triglyceride content rather than its density^{126 128} while others have come to the opposite conclusion.¹²⁸ Some domains of apoB appear more susceptible to conformational change than others; for example, Liu et al¹²⁶ found that as LDL density increased from 1.030 g/ml to 1.060 g/ml, there was increased expression of an epitope near the receptor binding site, but no detectable variation in the amino-terminal region of the protein. In addition to variation in the lipid content and protein structure, the sialic acid and neutral carbohydrate content of particles has been shown to be related to LDL size. Interestingly, it was the lipid bound rather than protein bound carbohydrate that exhibited the greatest difference.¹²⁹

Examination of whole LDL from normal and hypertriglyceridemic subjects revealed that LDL isolated from the latter were less able to bind to receptor on cultured cells, an effect attributed to altered apoB structure.^{127 130} Within the normal population, Swinkels at al¹³¹ showed that as LDL density decreased so did the ability to bind to Hep G2 cells and promote cholesterol esterification. Three further studies^{122 132 133} found that intermediate-sized LDL (LDL-II) exhibited the highest affinity for the LDL receptor while both lighter and denser LDL had about 30% lower affinity. Again, variation in receptor binding ability was linked to change in apoB structure rather than apoprotein or lipid composition. Reduced LDL-receptor interaction in small, dense LDL is consistent with the metabolic model developed above, ie, LDL with a prolonged residence time are more likely to be converted to a smaller species. However, it may also be the case that reduction in size leads to reduced binding (but size and receptor affinity are not inextricably linked).^{122 132} Campos et al¹²² noted a difference in LDL-I binding activity between pattern A and B subjects with the latter showing higher affinity. This was attributed to the higher apoE content of large LDL in pattern B. Few turnover studies have been conducted with LDL subfractions. Teng at al¹³⁴ found that a denser fraction was removed more slowly from the circulation than its lighter counterpart, consonant with the in vitro properties described above. The presence of "discrete" subpopulations in the LDL density range as evidenced by banding patterns on a salt gradient or gge raises the question as to whether conformational changes in apoB as well as affecting lipoprotein function may play a role in defining particle diameter, ie, there may be a limited number of stable states for the lipoprotein. This would be consistent with a threshold effect for the LDL-II to LDL-III transition.

	VLDL and LDL Heterogeneity and Atherosclerosis

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
Most individuals who succumb to CHD do not have frank hyperlipidemia but rather moderate disturbances in their plasma lipid profile. A full discussion of the role of ALP and small, dense LDL in promoting atherosclerosis is out of the scope of the present review but is dealt with in recent articles.^{68 80} Small, dense LDL appears to be a particularly atherogenic lipoprotein species. It is oxidized more easily than its larger counterparts.^{135 136} Further, it has been recently shown that LDL from subjects with an ALP binds more readily to arterial wall proteoglycans¹³⁷ thus potentially increasing the residence time of the lipoprotein in the artery wall. However, it should be remembered that the moderate hypertriglyceridemia of ALP is associated not only with small dense LDL, as described here, but also the presence of long-lived remnants in VLDL and IDL (Fig 1), both of which are believed to contribute to increased risk of CHD. Thus VLDL₁, if it fails to be cleared efficiently from the circulation, may spawn a cascade of atherogenic lipoproteins. In addition, raised fasting triglyceride levels are a determinant of slow chylomicron clearance and those with an ALP also accumulate chylomicron remnants in the bloodstream.⁸⁵ Understanding the mechanism of generation of the ALP will lead to development of drugs that can correct the underlying disturbance and it is predictable that their effects on the presentation of disease may be additive to that of existing agents.

	Selected Abbreviations and Acronyms

 

CHD = coronary heart disease IDL = intermediate density lipoprotein ER = endoplasmic reticulum LpL = lipoprotein lipase HL = hepatic lipase FH = familial hypercholesterolemia LRP = LDL receptor related protein gge = gradient gel electrophoresis CETP = cholesteryl ester transfer protein FCR = fractional catabolic rate

	Acknowledgments

 
We are indebted to Nancy Thomson for her excellent secretarial help in preparing this review. Both current and past colleagues made major contributions to the work performed in our laboratory that was supported by the British Heart Foundation and the Medical Research Council.

Received March 3, 1997; accepted May 8, 1997.

	References

Top Abstract Introduction Structure and Function of... Properties of VLDL Subfractions... Properties of VLDL Subfractions... Heterogeneity of Intermediate... LDL Structural and Metabolic... LDL Particle Size and... LDL Particle Size and... LDL Heterogeneity and CETP Genetic Influences on LDL... Metabolic Heterogeneity of LDL Linking VLDL and LDL... Linking Structural and... Properties of LDL Subfractions VLDL and LDL Heterogeneity... References

 
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