Atherosclerosis, 83 (1990) 59-67 Elsevier Scientific Publishers Ireland.

ATHERO

59 Ltd.

04491

Rapid isolation of low density lipoprotein (LDL) subfractions from plasma by density gradient ultracentrifugation Bruce A. Griffin, Muriel J. Caslake, Brigitte Yip, Graeme Christopher J. Packard and James Shepherd Institute oj Biochemistty,

(Revised,

W. Tait,

Royal Infirmary, Glasgow G4 OSF, Scotland ((1. K.)

(Received 12 July, 1989) received 1 December, 1989 and 5 March, (Accepted 12 March, 1990)

1990)

Summary High resolution density gradient ultracentrifugation (DGUC) and non-denaturing gradient gel electrophoresis (GGE) indicate that low density lipoprotein (LDL) in both normal and hyperlipidaemic subjects is composed of overlapping particle populations. A new centrifugation procedure has been developed which permits the separation of LDL subspecies directly from plasma within 24 h. The profiles obtained were analogous to those seen on gradient gel electrophoresis. LDL was divided into 3 fractions. The plasma concentration of LDL-I seen in young females was twice that in men (85.6 k 28.8 vs. 42.3 k 25.7 mg/dl, P < 0.005). LDL-II was not significantly different in any group while LDL-III was specifically elevated in coronary artery disease (CAD) patients (207.1 f 92.6 mg/dl in CAD vs. 87.4 + 79.6 mg/dl in normal men, P < 0.05). The presence of small, dense LDL detected either by density gradient centrifugation or gel electrophoresis was associated with raised triglyceride (TG) and low high density lipoprotein (HDL) cholesterol and may be a risk marker for coronary artery disease.

Key words:

Low density lipoprotein electrophoresis; Coronary

subfractions; Density artery disease

Introduction Low density lipoprotein (LDL), once considered a structurally and metabolically homogeneous entity, has now been shown not only to exhibit inter-individual heterogeneity but also to

___Correspondence to: Dr. B.A. Griffin, Institute istry, Royal Infirmary, Glasgow G4 OSF, U.K.

0021-9150/90/%03.50

0 1990 Elsevier Scientific

of Biochem-

Publishers

Ireland.

gradient

ultracentrifugation;

Gradient

gel

exist in multiple forms in the plasma of normal and hyperlipidaemic subjects [l-8]. The best characterised structural alteration in LDL is found in individuals with hypertriglyceridaemia. This is thought to be the result of a process of neutral lipid exchange first described by Nichols and Smith [9]. Here the elevated plasma levels of chylomicrons and very low density lipoproteins (VLDL) lead to increased transfer of triglyceride to the denser lipoproteins (LDL) and high density Ltd.

60 lipoprotein (HDL) via the agency of cholesteryl ester transfer protein (CETP). In exchange, cholesteryl ester is transferred into the triglyceride-rich lipoprotein. The modified LDL core becomes susceptible to lipolysis by hepatic and lipoprotein lipases and the action of these enzymes produces the smaller, denser particles that are characteristically found in hypertriglyceridaemics [lO,ll]. In fact, there is a negative correlation between plasma triglyceride levels and LDL size and flotation rate [12]. This remodelling provides an explanation for much of the between-subject variation in the lipoprotein’s structure but fails to explain the observations made some time ago in hyperlipidaemic patients [2,3] and recently in normals [5-7,12,13] that LDL is composed of discrete subfractions. These have been detected by the use of high resolution techniques such as density gradient centrifugation and non-denaturing gradient gel electrophoresis which show the presence of a limited number of LDL species differing in size and density. Subfractions of human LDL (d = 1.019-1.063 g/ml) prepared by the method of density gradient centrifugation described by Krauss et al. [6,7], yield up to four discernible classes, designated LDL-I-LDL-IV, although other methods have revealed multiple fractions [S]. In most healthy subjects LDL-II is the main component. Men have a

TABLE 1 DISTRIBUTION OF NORMAL AND HYPERLIPIDAEMIC SUBJECTS BETWEEN 4 PLASMA LIPID CATAGORIES AND LDL SUBFRACTION PATTERN No. of subjects expressed as a percentage of lipid category. LDL subfraction pattern A I= LDL-I primary peak ( I> 26.0 nm); A2 = LDL-II primary peak ( 2 25.5 nm); B3 = LDL-III primary peak ( < 25.5 nm); B4 = LDL-III primary peak ( < 24.7 nm). TG (mmol/I)

CH (mmol/I)

2 2

15 (36)

higher proportion of LDL-III, the dense subfraction, while females have significantly higher levels of LDL-I 1131. Little is known of the physiological significance of these subfractions or their relationship to coronary heart disease (CHD), although Austin and Krauss [12,13] have provided evidence that a preponderance of small, dense. LDL may be associated with an increased risk of CHD. In addition, they have shown that the LDL subfraction profile may in part be genetically determined. Family studies indicate the existence of an inherited syndrome of raised triglyceride, low HDL cholesterol and small, dense LDL [12]. This may be linked to the lipoprotein disorders known as hyperapobetalipoproteinaemia and familial combined hyperlipidaemia [l 7-- 161. The present study further examines the LDL subfraction distribution in normal and hyperlipidaemic subjects and describes a new density gradient centrifugation procedure for the rapid separation of discrete LDL fractions directly from plasma. Materials and methods Subjects

An initial survey of the gradient gel electrophoretic pattern of LDL subfractions was conducted in a mixed population of hyperlipidaemic (plasma cholesterol > 6.5 mmol/l, plasma triglyceride > 2.0 mmol/l, n = 84) and normolipidaemic (n = 59) subjects that were recruited from patients attending a Lipid Clinic and from healthy, laboratory staff respectively. The hyperlipidaemic group was subdivided on the basis of plasma triglyceride and cholesterol values (i.e. hypertriglyceridaemia = TG values above 2.0 mmol/l (cholesterol < 6.5 mmol/l) hypercholesterolaemia = cholesterol values above 6.5 mmol/l (TG < 2.0 mmol/l) and mixed hyperlipidaemia) (Table 1). Following these preliminary observations, the density, particle size and plasma concentration of LDL subfractions were determined in well defined. non age-matched subject groups of normal males (n = 7) and females (n = 7) and male patients with angiographically-proven coronary artery disease (n = 7). Normal volunteers were again recruited from laboratory staff. None suffered from hepatic, endocrine, renal or cardiovascular disease nor were they taking any medica-

61 TABLE

2

AGE AND PLASMA CONCENTRATION AND CAD PATIENTS

OF LIPID AND LIPOPROTEINS

IN NORMAL

FEMALE

AND MALE SUBJECTS

CAD = coronary artery disease. Mean values (mmol/l) f SD. Female (n = 7)

MaIe(n=7)

CAD (n = 7)

Age (yrs)

28f7

32*3

Range

18-37

26-35

54+5 46-59

Total cholesterol Triglyceride VLDL cholesterol

4.51+ 0.32 0.71+ 0.22 * 0.41+ 0.18 * * *

LDL cholesterol HDL cholesterol

2.32 + 0.26 1.77 +0.3s *

4.86 f 1.06 1.33*0.64 0.69*0.50 2.88 f 0.86 1.29 f 0.23

+ + +++ ++ +

7.17 + 2.03 + 0.89 + 5.50+ 0.99 *

2.30 0.35 0.30 2.00 0.22

++ +++ +++ ++ +++

Female vs. male: * P i 0.05, * * * P < 0.001; male vs. CAD; + P < 0.05, ++ P c 0.01, +++ P i 0.001; CAD vs. female; ” P < 0.01, “’ P < 0.001.

tion. All were within 20% of their ideal body weight and only one (M.K.) was a smoker. The diseased patients all had at least 2 vessels with > 50% stenosis and had stopped smoking 6 months prior to blood sampling. None of these subjects were obese. Two (B.E., B.O.) with non-familial, polygenic hyperlipidaemia, were undergoing weekly LDL-apheresis sessions and blood for subfraction analysis was withdrawn immediately before the apheresis procedure was begun i.e. when plasma lipoproteins were close to their pre-treatment, steady-state levels. At the time of examination none of the coronary group were being treated with lipid-lowering drugs or diets. The lipid and lipoprotein levels for the subjects are presented in Table 2. Gradient gel electrophoresis LDL was isolated from 4 ml of fasting plasma by sequential centrifugation at 40000 rpm at densities of 1.019 and 1.063 g/ml in a 40.3 Beckman fixed angle rotor [17]. The particle size distribution in this LDL preparation was analysed by non-denaturing gradient gel electrophoresis as described by Nichols et al. [18]. Briefly, 5 ~1 (approx. 5-10 pg protein) was applied to the top of a 2-16% gradient gel (Pharmacia, U.K.). Electrophoresis was performed for 24 h at 120 V according to the manufacturer’s instructions. The buffer used in the anodic and cathodic reservoirs was

0.09 M Tris/O.OS M boric acid/2.5 mM EDTA, pH 8.3. After separation the gel was fixed for 30 min in sulphosalicylic acid (lo%, w/v), stained with coomassie blue (O.l%, w/v) in methanol/ acetic acid/water (4: 1: 5, v/v) for 1 h and scanned with a Bio-Rad Model 620 Video Densitometer (Bio-Rad Laboratories Ltd., U.K.). The patterns obtained could be grouped into the ‘A’ and ‘B’ types of predominantly large or small LDL as described by Austin and Krauss [12] but this resulted in a loss of information from both the normal and patient groups. Instead, patterns A and B were subdivided into Al, A2 and B3 and B4 to provide more detail for analysis (pattern Al = LDL-I as a primary peak, particle diameter > 26.0 nm; A2 = LDL-II as a primary peak, > 25.5 nm; pattern B3 = LDL-III as primary peak < 25.5 nm; B4 = LDL-III as primary peak < 24.7 nm; primary in each case indicates the peak occupying the greatest area in the profile. Lipid and lipoprotein measurements were performed by the standard Lipid Research Clinics Protocol [17]. The concentration of cholesterol, triglyceride, free cholesterol and phospholipid was determined by enzymatic, calorimetric assays using commercially available reagents (BCL, Bell Lane, Lewes, East Sussex, U.K.; Cat. Nos. cholesterol, 816302; triglyceride, 816370; free cholesterol, 310328; phospholipid, 691844). LDL protein was determined by the method of Lowry et al. [19].

62 Fraction

Collector

j

Infusion

Fig. 1. Density

Pump

gradient and elution apparatus of LDL subfractions.

for the isolation

Density gradient ultracentrifugation A discontinuous salt gradient was devised which permitted the rapid isolation from plasma of LDL subfractions. Plasma from blood collected into dipotassium EDTA (1 mg/ml) was adjusted to a density of 1.09 g/ml by the addition of solid KBr (0.38 g per 3 ml plasma). The sample and 6-step salt gradient (Fig. 1) were introduced sequentially into polyvinyl alcohol-coated [20] polyallomer SW-40 tubes by peristaltic pump. The gradient was prepared and centrifugation carried out at a temperature of 23” C. All densities were checked using a digital densitometer (Paar Scientific Ltd., U.K.). The rotor was accelerated to 170 rpm over 4 min in a Beckman L8-60 ultracentrifuge and then centrifuged at 40000 rpm for 24 h. On completion of the run the rotor was stopped with the brake off. The gradient containing the separated LDL fractions was displaced upwards from the tube in an apparatus similar to that devised by Groot et al. [21]. A dense, hydrophobic material

(Maxidens, 1.9 g/ml. Nyegaard Ltd.). was introduced under the plasma layer by a constant infusion pump (Sage Instruments, Orion Research Incarp., U.S.A.) at a flow rate of 0.69 ml/mm. The eluate was passed through a UV detector (MSE/Fisons, U.K.) and continuously monitored at 280 nm. Measurement of the gradient in tubes in which plasma was replaced by a 1.09 g/ml density solution showed it to be continuous and curvilinear with LDL banding in the density range 1.025-1.060 g/ml. During the 24-h period the LDL bands did not reach isopycnic equilibrium. The elution times of the first, least dense, LDL fraction and the appearance of plasma proteins were reproducible and provided references for the identification of LDL subfractions. The recovery of LDL protein was 102 + 14% (mean + SD). LDL fractions prepared by this procedure were free from contamination by plasma proteins as judged by polyacrylamide electrophoresis and immunological techniques. Major LDL subfractions were identified by peak maxima that occurred between hydrated density intervals of 1.025-1.034 g/ml (LDL-I), 1.034-1.044 g/ml (LDL-II) or 1.044-1.060 g/ml (LDL-III). Minor subfractions were distinguished as distinct transitions or shoulders in the absorbance profile. The concentrations of the individual lipoprotein subfractions were determined by proportioning the mass (i.e. the sum of the protein, cholesterol, cholesterol ester, triglyceride and phospholipid content) of total LDL (d = 1.019-1.063 g/mlj prepared by sequential centrifugation according to the areas under the density gradient absorbance profile. The detection system measured LDL concentration as absorbance at 280 nm and this was corrected to lipoprotein mass equivalence by applying specific extinction coefficients (LDL-I 1 optical density unit (1 OD) = 2.63 mg lipoprotein/ml, LDL-II 1 OD = 2.94 mg lipoprotein/ml, LDL-III 1 OD = 1.92 mg lipoprotein/ml). The stability of the LDL subfraction distribution was determined by comparing the subfraction profile obtained from fresh plasma (analysis begun within 24 h) with that of stored plasma (24 h, 4” C) and frozen plasma (223 weeks, - 20 o C). The effect of prolonged centrifugation was examined by comparing LDL subfractions from fresh plasma with those prepared from sequentially isolated LDL.

63

T

T ?

0j

rnm LDL

IlIla

Subfraction

III

In

Patterns

rn

III

(GGE)

Fig. 2. Relationship between LDL subfraction pattern by gradient gel electrophoresis (GGE) and the concentration of plasma triglyceride and HDL cholesterol (mmol/l). Pattern Al: primary peak = LDL-I (peak diameter > 26.0 mn, n = 30); pattern AZ: primary peak = LDL-II (peak diameter 2 25.5 nm, n = 75); pattern B3: primary peak = LDL-III (peak diameter < 25.5 nm; n = 12); pattern B4: primary peak = LDL-III (peak diameter < 24.7 nm, n = 26).

Reproducibility of the density gradient fractionation procedure was assessed by replicate analysis of single plasma samples. The coefficient of variation for LDL-I, II and III for six replicate samples (within-rotor) was 3.4%, 2.0% and 5.4%, respectively. To avoid the introduction of storage artefacts upon LDL structure, the examination of between-batch variation was restricted to the simultaneous centrifugation of 6 plasma samples in 2 rotors. In this comparison the between-batch variation for LDL-I, II and III was less than 6.5%. LDL subfractions were significantly correlated between rotors (r = 0.996 (I), 0.997 (II), 0.998 (III)). Statistical comparisons were performed using Student’s t-test for unpaired data and Pearson’s correlation coefficient. Results The four subfraction profiles (Al-B4) seen in our GGE-based survey of patients and normal subjects are shown in Fig. 2. Most normolipidaemic (56%) and hyperlipidaemic (SOW) subjects exhibited pattern A2 (LDL-II > I&III) fol-

lowed by pattern Al, predominantly larger LDL (LDL-I > II&III), in normals (29%) and pattern B4, small LDL (LDL-III > II) in hyperlipidaemia (26%). In the latter group the distribution of subjects with TG values > 2 mmol/l was displaced towards smaller LDL particles (46% with hypertriglyceridaemia and 36% with mixed hyperlipidaemia were pattern B4) irrespective of a plasma cholesterol of > 6.5 mmol/l. In addition, the distribution of subjects with cholesterol > 6.5 mmol/l was only displaced towards larger LDL particles in the absence of a raised TG (38% with hypercholesterolaemia were pattern Al) (Table 1). Overall, there were significant associations between the concentrations of plasma triglyceride and HDL cholesterol (r = - 0.37, P < O.OOl), triglyceride and LDL particle size (r = - 0.60, P -c 0.0001) and HDL cholesterol and LDL particle size (r = 0.44, P < 0.005). Of subjects with pattern Al, 87% had an HDL cholesterol above 1.3 mmol/l. The concentrations of LDL and total plasma cholesterol were unrelated to the distribution of LDL particle size (Table 1). In this initial screen plasma triglyceride was positively correlated with age (r = 0.3, P -C 0.02) but the latter was unrelated to LDL subfraction pattern. The relationships between the pattern of LDL subfractions, triglyceride and HDL cholesterol are represented in Fig. 2. The density gradient developed for the separation of LDL subfractions directly from plasma consistently resolved 3 subfractions which corresponded in density and particle size to LDL-I to LDL-III described by Krauss and Burke [7]. The relationship between density and particle size profiles of representative male and female subjects is illustrated in Fig. 3. Interestingly, the density interval for the small, dense LDL-III subfraction often extended into that defined for LDL-IV [13], a subfraction that was detectable in trace amounts only by electrophoresis. However, the peak diameter for LDL isolated from this density region fell within that defined for LDL-III. In general, density centrifugation and gel electrophoresis appeared to have complementary resolving capacities at either end of the LDL spectrum, LDL-I being more effectively distinguished on a basis of density and LDL-III on the basis of size. Overall, the resolution of the density gradient procedure

64

3GE -

DGUC

Kh

Particle Diameter I I to34

1.044

Density

(gbl

(nm)

J

1060

CAD patients compared with both normal groups (Table 3). These quantitative differences derived from density gradient absorbance profiles are supported by the demonstration on gradient gel electrophoresis of larger LDL peak diameters in females relative to males (P < 0.05) and in males relative to CAD patients (P < 0.05). On closer inspection of the subfraction profiles (Fig. 5) the differential resolving capacities of the two procedures become apparent. LDL-I was effectively resolved from LDL-II by density (M.U., B.D., D.E., T.D.), and LDL-II from LDL-III by particle size (P.E., M.Y., M.E., T.H.). The lipid and lipoprotein profiles indicated, as expected, higher concentrations of apoB-containing lipoproteins (VLDL, LDL) and triglycerides in the male and CAD groups and significantly higher concentrations of HDL in the female group (Table 2). Plasma triglyceride was positively correlated with LDL-III concentration but not LDL-I or LDL-II (Fig. 6). LDL-I and LDL-III were inversely related (r = -0.55, P -c 0.05) while LDL-I was correlated with HDL cholesterol (r = 0.76, P -c 0.01). On gel electrophoresis the peak LDL

)

Fig. 3. Relationship between the density (DGUC) and particle size (GGE) of LDL subfractions in a single female and male subject.

was more reproducible and it was used for quantification of the individual subfractions. Storage of plasma at 4°C for 24 h and at - 20 o C for 2-3 weeks resulted in a loss of resolution between LDL-I and LDL-II due to a loss (possibly denaturation) of lighter LDL particles. This change was accompanied by a small increase in denser, LDL-III particles (Fig. 4). A similar change was observed when LDL prepared by sequential centrifugation (over 48 h) was subjected to density gradient ultracentrifugation. The distribution profiles of LDL density and particle size varied markedly within and between the groups. Higher levels of LDL-I and LDL-III were characteristic of the female and CAD groups respectively (Fig. 5). The concentration of LDL-I was significantly higher in females compared with males and CAD patients, whereas the concentration of LDL-III was significantly higher in the

--f r.“l&5

1.634

Density

1.644

l&

(g/ml)

Fig. 4. Effect of storage and prolonged centrifugatlon upon LDL subfraction profile. LDL subfraction profiles prepared by the density gradient centrifugation of: (a) and (d) fresh plasma, within 24 h: (b) LDL (d = 1.019-1.063) derived from plasma (a); (c) plasma (a) stored for 24 h at 4O C; (e) plasma (d) stored for 2 weeks at - 20’ C; (f) plasma (d) stored for 3 weeks at -2OOC.

65 LDL Size Profiles (peak diameter-nm) Females

Males

CAD

LDL

Males

, TotalLDL

(mg lipqxotein/l~d~

Fig. 6. Correlation of total LDL and LDL subfraction mass with plasma triglyceride. LDL subfractions of certain subjects were either undetectable or could not be reliably quantified.

LDL Density Profiles (g/ml) Females

Subfractions

CAD

diameter was correlated positively with HDL cholesterol (r = 0.80, P -e0.001) and negatively with triglyceride (r = - 0.83, P < 0.001).

Fig. 5. Distribution profiles of LDL size and density in normal females and males and CAD patients.

TABLE

3

CONCENTRATION OF LDL SUBFRACTIONS IN NORMAL FEMALE AND MALE SUBJECTS AND CAD PATIENTS, AS DETERMINED BY DENSITY GRADIENT SEPARATION CAD = coronary artery disease. protein/100 ml plasma) f SD.

LDL-I LDL-II LDL-III

Mean

values

(mg

lipo-

Female ( n = 7)

Male (n = 7)

CAD (n = 7)

85.6 f 28.8 * * 109.9 + 29.6 31.4rt 14.2

42.3+ 25.7 166.9 f 108.5 87.4+ 79.6 +

32.3 + 35.9 ++ 183.9 f 97.6 207.1 rt 92.6 ++

Female vs. male: * * P < 0.05; male vs. CAD, + P < 0.05; CAD vs. female, ” P < 0.05.

It has been known for some years that LDL in hyperlipidaemic subjects shows marked polydispersity [l-4] but it is only recently that this phenomenon has been described in subjects with relatively normal plasma lipid levels. Non-denaturing gel electrophoresis has a high resolving capability for both HDL and LDL and it was the demonstration of multiple LDL species on gradient gels that led to the wide recognition that this lipoprotein is composed of a small number of discrete but overlapping particle populations. Our initial screen of LDL subfraction pattern in normal and hyperlipidaemic subjects yielded significant, qualitative associations between the distribution of LDL particle size and the concentration of plasma triglyceride and HDL cholesterol. These associations were strengthened by the subdivision of pattern A and B into Al, A2, B3 and B4 (Table 1, Fig. 2) but the electrophoretic separation proved to be, in the main, inadequate for a quantitative assessment of LDL subfraction concentrations. The density gradient procedure described in this publication yielded profiles that not only closely resembled those seen on gel electrophoresis but also allowed the estimation of lipoprotein subfraction plasma concentrations. The 2 methods were found to be complementary in that in most cases the centri-

66 fugal procedure was superior at separating LDL-I and LDL-II while LDL-II and LDL-III were more effectively resolved by electrophoresis. In developing the density gradient method we evaluated previously published techniques and found that those which employed the fractionation of sequentially isolated LDL showed a diminution of larger LDL species and an increase in smaller denser particles (Fig. 4). We have also noted that storage of plasma at 4 o C for only 24 h leads to noticeable changes in the subfraction profile, usually again an increase in LDL-III. Hence, in this study centrifugal separation was performed on fresh plasma as soon as possible (within 24 h of venipuncture) to minimise artifactual changes. The existence of multiple LDL species and their variability between subjects can be explained only in part by neutral lipid transfer via CETP. It is difficult to see how simple core exchange can produce 3-4 discrete species in a single subject. They must arise either from different synthetic sources or be generated in the circulation by lipolytic processes that give rise to separate, stable metabolic end-products. The existence of metabolically distinct species is supported by LDL turnovers in humans [22]. Quantitation of LDL subfraction concentrations in this study revealed a number of potentially important relationships. LDL-I was higher in females than in males (Table 3) and was correlated with HDL cholesterol. This observation may be attributed to the lower hepatic lipase levels found in women [23]. This enzyme appears to be required for the conversion of intermediate density lipoprotein to LDL [24] and LDL-I may represent a transient step in this delipidation cascade. The plasma concentration of LDL-II did not vary significantly between the groups studied, nor was it related to triglyceride or HDL levels. On the other hand LDL-III was increased in men versus women and was high in the CAD group. Furthermore, its concentration was positively correlated with triglyceride in the wider survey of normal and hyperhpaemic subjects (Fig. 2) and in the smaller groups studied by both separation techniques (Fig. 6). This agrees well with previous findings [12,25,26]. Consideration of the gel electrophoresis profiles alone (Fig. 2) might suggest that LDL-III arose primarily by remodelling of LDL-I and LDL-II via CETP ac-

tivity so that the concentration of the most dense fraction increased at the expense of the two less dense species. However, we observed no diminution of LDL-I and LDL-II at higher plasma triglycerides (Fig. 6). Rather the association of high LDL-III with raised plasma triglyceride appeared to be linked to an elevation in total LDL. It is possible that this LDL species derived from increased VLDL synthesis in subjects with raised triglyceride levels. This in turn gives rise to increased LDL production. Acknowledgements We thank Patricia Price for excellent secretarial help. This work was support by a grant from the British Heart Foundation (187006). References 1 Lindgren. F.T.. Jensen, L.C., Wills, R.D. and Freem, N.K., Flotation rates, molecular weight and hydrated densities of the low density lipoproteins, Lipids, 4 (1969) 337. 2 Hammond, M.G. and Fisher, W.R., The characterisation of discrete series of low density lipoproteins in the disease hyper-preB lipoproteinemia. J. Biol. Chem., 246 (1971) 5454. 3 Hammond, M.G., Mengel, M.C., Warmke, G.L. and Fisher, W.R., Macromolecular dispersion of human plasma low density lipoproteins in hyperlipoproteinemia. Metabolism, 26 (1977) 1231. 4 Fisher. W.R.. Heterogeneity of plasma low density lipoproteins. Manifestations of the physiologic phenomenon in man. Metabolism, 32 (1983) 283. 5 Krauss, R.M., Lindgren. F.T. and Ray, R.M., Interrelationships among subgroups of serum lipoproteins in normal human subjects, Clin. Chim. Acta, 104 (1980) 275. 6 Shen, M.M.S.. Krauss, R.M., Lindgren, F.T. and Forte, T.M.. Heterogeneity of serum low density lipoproteins in normal human subjects, J. Lipid Res., 22 (1981) 236. 7 Krauss, R.M. and Burke. D.J., Identification of multiple subclasses of plasma lipoproteins in normal humans, J. Lipid Res., 23 (1981) 97. 8 Chapman, M.J., Laplaud, P.M., Luc. G., Forgez, P.. Bruckert, E., Goulinet, S. and Lagrange, D., Further resolution of the low density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation, J. Lipid Res., 29 (1988) 442. 9 Nichols, A.V. and Smith, L.. Effect of very low density lipoproteins on lipid transfer in incubated serum. J. Lipid Res., 6 ( 1965) 206. 10 Eisenberg, S., Gavish, D.. Oschry, Y., Fainaru, M. and Deckelbaum, R.J., Abnormalities in very low, low. and high

67

11

12 13

14

15

16

17 18

19

density lipoproteins in hypertriglyceridemia. Reversal towards normal with bezafibrate treatment, J. Clin. Invest., 74 (1984) 470. Brunzell, J.D., Albers, J.J., Chait, A., Grundy, S.M., Groszek, E. and McDonald, G.B., Plasma lipoproteins in familial combined hyperhpidemia and monogenic familial hypertriglyceridemia, J. Lipid Res., 24 (1983) 147. Austin, M.A. and Krauss, R.M., Genetic control of low density lipoprotein subclasses, Lancet, ii (1986) 592. Mushner, T.A. and Krauss, R.M., Lipoprotein subspecies and risk of coronary disease, Chn. Chem., 34/8B (1988) B78. Austin, M.A., Breslow, J.L., Hennekens, C.H., Buring, J.E., Willet, W.C. and Krauss, R.M., Low density lipoprotein subclass patterns and risk of myocardial infarction, JAMA. 260 (1988) 1917. Teng, B., Thompson, G.R., Sniderman, A.D., Forte, T.M., Krauss, R.M. and Kwiterovich, P.O., Jr., Composition and distribution of low density lipoprotein fractions in hyperapobetalipoproteinemia, Proc. Natl. Acad. Sci. USA, 80 (1983) 6662. Sniderman, A.D., Shapiro, S., Marpole, D., Skinner, B.. Teng, B. and Kwiterovich, P.O., Jr., Association of coronary arteriosclerosis with hyperapobetalipoproteinemia (increased protein but normal cholesterol levels in human plasma low density lipoproteins), Proc. Natl. Acad, Sci. USA, 77 (1980) 604. Lipid Research Clinics Program Manual of Laboratory Operations, DHEW Publication No. (NIM) 75 (1975). Nichols, A.V., Krauss, R.M. and Musliner, T.A. Non-denaturing polyacrylamide gradient gel electrophoresis, Methods Enzymol., 128 (1986) 417. Lowry, O.H., Rosebrough, N.J., Fat-r, A.L. and Randall, R.J., Protein measurement with the Fohn phenol reagent, J. Biol. Chem., 193 (1951) 265.

20 Hohnquist, L., Surface modification of Beckman Ultraclear centrifuge tubes for density gradient centrifugation of lipoproteins, J. Lipid Res., 23 (1982) 1249. 21 Groot, P.H.E.., Scheek, L.M., Havekes, L., Noort, W.L. and van? Hooft, F.M., A one-step separation of human serum high density lipoproteins 2 and 3 by rate-zonal density gradient ultracentrifugation in a swinging bucket rotor, J. Lipid Res., 23 (1982) 1342. 22 Foster, D.M., Chait. A., Albers, J.J.. Failor, A., Harris, C. and Brunzell, J.D.. Evidence for kinetic heterogeneity among human low density lipoproteins, Metabolism, 35 (1986) 685. 23 Applebaum-Bowden, D., Haffner, S.M.. Wahl, P.W., Hoover, J.J., Wamick, G.R., Albers, J.J. and Hazzard, W.R., Post-heparin plasma triglyceride lipases. Relationships with very low density lipoprotein triglyceride and high density lipoprotein, cholesterol, Arteriosclerosis, 5 (1985) 273. 24 Demant, T., Carlson, L.A., Holmquist, L., Karpe. F., Nilsson-Ehle, P., Packard, C.J. and Shepherd, J., Lipoprotein metabolism in hepatic lipase deficiency studies on the turnover of apoprotein B and on the effect of hepatic lipase on high density lipoprotein, J. Lipid Res., 29 (1988) 1603. 25 McNamara, J.R., Campos, H., Ordovas, J.M., Peterson, J., Wilson, P.W.F. and Schaefer, E.J.. Effect of gender, age and lipid status on low density lipoprotein subfraction distribution. Results from the Framingham Offspring Study, Arteriosclerosis, 7 (1987) 483. 26 Crouse, J.R., Parks, J.S., Schey, H.M. and Kahl. F.R., Studies of low density lipoprotein molecular weight in human beings with coronary artery disease. J. Lipid Res., 26 (1985) 566.