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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:794-801.)
© 1996 American Heart Association, Inc.

Articles

Differences in Receptor Binding of LDL Subfractions

Hannia Campos; Kay S. Arnold; Maureen E. Balestra; Thomas L. Innerarity; Ronald M. Krauss

From the Donner Laboratory, University of California, and the Department of Molecular and Nuclear Medicine, Life Sciences Division, Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley (H.C., R.M.K.), and the Gladstone Institute of Cardiovascular Disease, San Francisco (K.S.A., M.E.B., T.L.I.), California.

Correspondence to Ronald M. Krauss, MD, Ernest Orlando Lawrence Berkeley National Laboratory, University of California, Donner Laboratory Room 465, One Cyclotron Rd, Berkeley, CA 94720. E-mail rmkrauss@lbl.gov.

	Abstract

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Abstract Differences in low density lipoprotein (LDL) receptor-binding affinity among LDL particles of different size were examined in competitive binding assays in human skin fibroblasts and LDL (d=1.020 to 1.050 g/mL) from subjects with a predominance of large (272 Å), medium (259 to 271 Å), and small (257 Å) LDL. Among 57 normolipidemic subjects with LDL cholesterol (-C) levels <160 mg/dL, binding affinity was reduced by 16% in those with predominantly large LDL and by 14% in those with small LDL compared with most subjects who had a predominance of medium-size LDL and in all LDL size subgroups in 66 subjects with LDL-C 160 mg/dL. Differences in LDL receptor-binding affinity were further investigated by using LDL density subfractions (I, d=1.026 to 1.032 g/mL; II, d=1.032 to 1.038 g/mL; and III, d=1.038 to 1.050 g/mL) from three subjects with predominantly large (pattern A) and small (pattern B) LDL particles. The binding affinity (K_d) of LDL-II was similar for patterns A and B (9.2±1.4 and 9.4±0.7, respectively) and 30% lower in LDL-III from both groups (P<.05). The binding affinity of LDL-I in pattern A (12.6±1.5 µg/mg) was lower (P<.05) than that in LDL-II and LDL-I from pattern B (8.0±2.4 µg/mg). After incubation with a monoclonal antibody that specifically blocked the LDL receptor-binding domain of apoE, LDL-I from two pattern B subjects showed substantially lower binding affinity (K_d=20.0 and 19.2 µg/mg) than in pattern A (K_d=13.2 and 14.2 µg/mg), a result consistent with our finding of a higher apoE content in pattern B LDL-I (P<.001). Thus, factors associated with variations in particle size and apoE content in LDL subclasses in normolipidemic subjects contribute to the differences in LDL receptor binding that may result in differing metabolic behavior in vivo.

Key Words: LDL subclasses • LDL size • apoE • LDL receptor • cholesterol

	Introduction

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Several studies have shown that distinct subpopulations of LDL particles differ in size, density, and composition.^{1 2 3} Seven LDL size species have been identified within four density subgroups on the basis of the distribution of LDL size as determined by GGE.^{3 4 5 6} In most individuals the LDL subclass pattern can be categorized as pattern A or pattern B.⁶ Pattern A is characterized by the predominance of larger, more buoyant LDL particles (diameter >255 Å), including LDL-I (d=1.025 to 1.032 g/mL) and LDL-II (d=1.032 to 1.038 g/mL). Pattern B is characterized by the predominance of small LDL particles (diameter 255 Å), including LDL-III (d=1.038 to 1.050 g/mL) and lesser amounts of LDL-IV (d=1.050 to 1.063 g/mL).^{5 6} Pattern A is more common in randomly selected populations, with 90% of women and 67% of men having this pattern.³ Pattern B is associated with increased TG and reduced HDL-C levels and an increased risk for CAD,^{3 7 8 9 10} and family studies indicate that pattern B is a genetically influenced trait.^{6 11} Further investigations have suggested linkage of the gene for LDL particle size with a locus on chromosome 19 near the LDLR gene¹² as well as with other loci on chromosomes 11, 16, and 6.¹³

Differences in the receptor-binding affinity of the various LDL subfractions have been reported, but the results have not been consistent.^{14 15 16 17 18} Swinkels et al¹⁴ found no differences in the receptor binding of LDL subfractions to LDLRs on fibroblasts and HepG2 cells, but two other studies found that both buoyant (d=1.024 to 1.037 g/mL) and dense (d=1.036 to 1.047 g/mL) LDL subfractions had reduced binding affinity compared with medium-density LDL subfractions (d=1.027 to 1.041 g/mL).^{16 18} Still other studies have found greater binding affinity for buoyant LDL subfractions (d=1.024 to 1.033 g/mL) compared with both medium and dense LDL subfractions (d=1.028 to 1.045 g/mL).^{15 17} Some of the disparity in results may be due to the use of different cell lines, pooled plasma, and/or isolation of plasma lipoprotein subfractions from different density ranges. Moreover, there are no data on the receptor-binding affinity of whole-LDL preparations from subjects with different LDL particle size distributions as determined by GGE or on the binding affinity of isolated LDL subfractions from subjects with different LDL subclass patterns.

To examine whether differences in receptor-mediated LDL catabolism contribute to the differences in LDL subclass distribution in pattern A and pattern B subjects, we carried out a series of studies with the following objectives: (1) to test whether whole-LDL preparations (d=1.020 to 1.050 g/mL) from subjects with predominant LDL particle diameters (determined by GGE) corresponding to LDL-I, LDL-II, or LDL-III differ in receptor-binding affinity; (2) to test whether the binding affinities of LDL subfractions I, II, and III from subjects with pattern A differ among themselves and from equivalent fractions from pattern B subjects; and (3) to determine whether there are differences in ligand-binding affinity of LDLRs derived from pattern A versus pattern B subjects.

	Methods

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Study I: Comparison of LDLR Affinity of Whole-LDL Preparations From Subjects With Different LDL Subclass Distributions
Subjects
The study subjects were 72 men and 51 women who came to our clinic and did not have familial defective apoB-100 (as determined by normal binding affinity to the LDLR). Exclusion criteria included apoE 2/2 phenotype; use of lipid altering drugs, hormones, or antihypertensive medications; and TG concentrations >500 mg/dL. On the basis of National Cholesterol Education Program guidelines,¹⁹ the subjects were divided into two groups: (1) those with normal LDL-C concentrations (<160 mg/dL, n=57: 25 women and 32 men) and (2) those with high LDL-C concentrations (160 mg/dL, n=66: 26 women and 42 men). All subjects who participated in the three studies signed a consent form that had been preapproved by the Committee for the Protection of Human Subjects at our institution.

Specimen Collection and LDL Subclass Analysis
Blood samples were obtained by venipuncture after an overnight fast and collected in EDTA-containing tubes (final concentration, 0.15% wt/vol). Plasma samples were obtained by centrifugation and kept at 4°C for no more than 2 weeks for lipid and lipoprotein analyses. LDL particle diameters were determined from whole plasma by electrophoresis on nondenaturing Pharmacia 2% to 16% polyacrylamide gradient gels as previously described.^{1 20} The gels were stained with oil red O (Sigma Chemical Co) and scanned with a Transidyne RFT scanning densitometer (Transidyne Corp). LDL particle diameters were estimated from calibration curves that were constructed by using latex beads (Duke Scientific) and high-molecular-weight standards (Pharmacia). Subjects were grouped into three categories on the basis of their predominant LDL peak particle diameter and distribution cutpoints at the 25th and 75th percentiles. The three groups were defined as large- (272 Å), medium- (258 to 271 Å), and small- (257 Å) LDL groups. These categories are consistent with reported LDL subclass category size ranges.^{1 4 5 20}

Fibroblast Binding Assays
LDL (d=1.020 to 1.050 g/mL) fractions were prepared from each subject's plasma sample by using standard procedures^{21 22} and were radiolabeled by the ICl procedure.²¹ Binding affinity was determined from the concentration of unlabeled LDL (test lipoprotein) that was required to displace 50% of the ¹²⁵I-labeled LDL in foreskin fibroblast cultures from normal infants.²³ Fibroblast cell lines were maintained at 37°C in DMEM containing 10% fetal bovine serum and grown to confluence in 22-mm plastic wells; afterward, the LDLRs were upregulated by incubation for 48 hours with DMEM containing 10% LPDS and for 2 hours on ice²³ with 1 mL DMEM/HEPES/LPDS. A constant concentration of ¹²⁵I-labeled lipoprotein was used with increasing concentrations of unlabeled competing ligand. Wells that contained ¹²⁵I-labeled but not the unlabeled lipoproteins were used as the 100% control. The labeled 100% control sample predominantly consisted of LDL-II particles with a mean peak particle diameter of 262±2 Å. Wells that included ¹²⁵I-labeled lipoproteins and a 100-fold excess of unlabeled lipoproteins were used to determine the nonspecific binding of the ¹²⁵I-labeled lipoproteins. Values for the unlabeled test lipoproteins were expressed relative to the 100% control sample.

Study II: Comparison of LDLR Binding Affinity of LDL Subfractions From Normolipidemic Subjects With Subclass Patterns A and B
Subjects
Three subjects with pattern A and three with pattern B were selected for the second study. Selection criteria were otherwise the same as for Study I.

Specimen Collection and Lipoprotein Preparation
Blood samples (250 mL) were collected in EDTA (final concentration, 0.15%). Immediately after separation of the plasma we added merthiolate (final concentration, 0.25 mg/mL), aprotinin (23 µg/mL), penicillin (50 U/mL), streptomycin (50 µg/mL), and the antioxidant Trolox (2.5 µg/mL) to the plasma. LDL preparations (d=1.020 to 1.050 g/mL) were isolated by standard ultracentrifugation methods^{21 22} and then subfractionated by equilibrium density gradient ultracentrifugation in 7-mL tubes in a Beckman SW45 rotor as previously described,^{1 2} except that the following fractions were collected: 0.5 to 2.5 mL (d=1.026 to 1.032 g/mL; LDL-I), 2.5 to 3.5 mL (d=1.032 to 1.038 g/mL; LDL-II), and 3.5 to 5.5 mL (d=1.038 to 1.050 g/mL; LDL-III). Peak particle diameters were determined as described above for whole plasma, except that gels were stained for protein with Coomassie Brilliant Blue R250. The relative particle size distribution was determined for each LDL subfraction by calculating the total protein-stained area within each LDL size interval by densitometric scanning of 2% to 16% gradient gel areas.

Fibroblast Binding Assays
Direct binding assays at 4°C were carried out as previously described²³ using fibroblasts that were grown to confluence in 35-mm plastic wells after the LDLRs had been upregulated as described above. Cells were incubated for 5 hours on ice with 1 mL DMEM/HEPES/LPDS containing ¹²⁵I-labeled LDL subfractions. Each experiment included three LDL subfractions from a pattern A and three from a pattern B subject. Each experiment was performed twice, once with a cell line from a pattern A subject and once with a cell line from a pattern B subject from Study III (see below). However, because receptor binding did not significantly differ between cell lines, the reported binding affinity for each of the three LDL subfractions for each subject represents the mean of the two experiments. A control sample (d=1.020 to 1.050 g/mL) consisting predominantly of LDL-II particles (mean peak particle diameter, 263±3 Å) was included in every second experiment. Nonspecific binding was calculated by using a 100-fold excess of unlabeled control LDL and was defined as the amount bound in the presence of the 100-fold excess of unlabeled lipoprotein. Cellular protein content was determined by the method of Lowry et al.²⁴ Within-experiment cellular protein variation was <8% and between-experiment variation <16%. Scatchard analyses were used to determine equilibrium dissociation constant (K_d, in micrograms per milligram of cell protein) and the number of receptor-bound lipoproteins at receptor saturation (B_max, in nanograms of bound protein per milligram of cell protein).²³

The contribution of apoE to the binding of LDL-I subfractions was determined by using monoclonal antibody ID7 kindly provided by Dr Ross Milne. This antibody specifically inhibits binding of apoE to the LDLR.²⁵ Experiments were performed by incubating each test lipoprotein with a 10-fold excess (by protein mass) of antibody for 1 hour before the experiment. These experiments were carried out with LDL-I subfractions from two pattern A and two pattern B subjects.

Study III: Comparison of LDLR Function in Skin-Derived Fibroblast Cultures From Subjects With LDL Subclass Patterns A and B
Subjects
The subjects were 19 free-living, healthy, normolipidemic men who were participants in a previously described dietary intervention study.²⁶ They were not taking lipid-lowering drugs or antihypertensive medications, were nonobese, had total cholesterol concentration <95th percentile for their age decile, and had TG concentrations <500 mg/dL. The subjects were selected if they consistently expressed LDL subclass pattern A (n=10) or B (n=9) on both a high-fat (38% carbohydrate and 46% total fat) and a low-fat (60% carbohydrate and 24% total fat) diet that they consumed for 6 weeks each in a crossover design²⁶ and agreed to a skin biopsy.

Specimen Collection and Lipoprotein Preparation
Skin was obtained from the 19 participants by punch biopsy, and fibroblast cell lines were established and maintained as described previously.²³ Blood samples were obtained as described for Study I. Normal human LDL preparations (d=1.020 to 1.050 g/mL) from a human control subject with predominantly LDL-II particles (peak particle diameter, 261 Å) were isolated as described above. Cholesterolemic apoE HDL_c fractions (d=1.006 to 1.020 g/mL) were prepared and used for measurement of the specific binding of ¹²⁵I-labeled canine apoE HDL_c as previously described.²⁷

LDL Subclass Analysis
LDL particle diameters were determined for whole plasma by electrophoresis on nondenaturing Pharmacia 2% to 16% polyacrylamide gradient gels as described above. Classification of subjects with pattern A or B on both high-fat and low-fat diets was carried out by three independent observers as previously described.⁶

Fibroblast Binding Assays
Direct binding assays at 4°C were carried out as described for Study II. Cells were incubated on ice with either ¹²⁵I-labeled LDL (5 hours) or ¹²⁵I-labeled apoE HDL_c (7 hours). To reduce interassay variability each binding experiment included pattern A and pattern B cell lines. Growth-curve experiments were performed for all cell lines to match the growth characteristics of pattern A and B cell lines. The investigators remained blinded to the subjects' LDL subclass pattern (A or B) throughout the study. Three pattern A and three pattern B cell lines were tested in each experiment on at least three occasions for determination of LDL and apoE HDL_c binding, for a total of 13 experiments. Within-experiment cellular protein variation was <10% and between-experiment variation <22%.

Chemical Analysis
Plasma cholesterol and TG concentrations were determined enzymatically on a Gilford Impact 400E analyzer using Gilford reagents. Our laboratory participates in the Centers for Disease Control and Prevention standardization program. HDL-C was measured after heparin-manganese precipitation of apoB-containing lipoproteins.²⁸ Plasma apoB concentrations were measured by single radial immunodiffusion²⁹ by using plates and standards from Tago, Inc, over the range of 25 to 150 mg/dL (or higher, after dilution).

ApoE concentrations were measured on LDL subfractions by enzyme-linked immunosorbent assays using an antibody sandwich-style assay.³⁰ Purified goat anti-human apoE IgG (International Immunology Corp) was bound to the solid phase of 96-well microtiter plates (100 ng/well; C-Maxisorp plates, Nunc). The plates were washed with PBS and active sites were blocked with 2% BSA, 0.5% casein hydrolysate, and 0.1% Tween-20. Triplicate samples were applied to the plate in 1% BSA, 1% casein hydrolysate, and 0.375% Tween-20 in 100 µl per well. The plates were incubated at room temperature for 2 hours and then washed with PBS. Purified and biotin-labeled anti-human apoE IgG was then added to the wells, and after incubation for 1 hour the plates were washed again. Color development and quantitation of apoE were accomplished by addition of streptavidin-conjugated horseradish peroxidase and color substrate solution (o-phenylenediamine 2HCl in citric acid–phosphate buffer, pH 5.3). The color was allowed to develop for 20 minutes and stopped by addition of 3 mol/L HCI. Aliquots of frozen (-80°C) standard and reference plasma were analyzed for apoE concentration by Northwest Lipid Research Laboratories. Absorbance was measured at 490 nm and a standard curve was fitted to a four-parameter equation for calculation of unknowns. The linear portion (ie, working range) of the standard curve was 1 to 60 ng. Reference control plasma had intra-assay and interassay coefficients of variation of 6.5% and 9.5%, respectively. Assays were performed in duplicate or triplicate with standards, controls, and unknowns on the same plate and with a coefficient of variation of ±5%. The apoB to apoE molecular ratios were estimated by using molecular weights of 550 000³¹ and 34 000,³² respectively.

Statistical Analyses
Differences in plasma lipoproteins, receptor-binding affinity, B_max, and apoE to apoB molar ratio in Studies II and III were determined by paired and unpaired t test analyses. Pearson's correlation coefficients were used to study the association of binding affinity with age, plasma lipoprotein concentration, and LDL particle diameter. General linear models adjusting for age were used to study the effect of LDL-C and LDL particle size on binding affinity (Study I). All statistical analyses were performed with the Statistical Analysis System (SAS Institute, Inc).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Study I: LDLR Affinity of Whole-LDL Preparations From Subjects With Different Predominant LDL Subclass Patterns
Table 1 shows the LDL subclass binding-affinity comparisons (percent of the value for medium-size control LDL) in subjects with normal LDL-C concentrations (<160 mg/dL). Compared with the results from subjects with a predominance of medium-size LDL, binding affinity was significantly (P=.02) reduced by 16% in subjects with a predominance of large LDL and by 11% in subjects with a predominance of small LDL. However, as shown in Table 2, subjects with a high LDL-C concentration (160 mg/dL) showed similar receptor-binding affinity values for the three LDL size categories. Also, LDL binding affinities for large- and small-LDL categories in subjects with a high LDL-C level were higher when compared with those for large (20%, P<.07 and 23%, P<.05, respectively) and small (13%, P<.09 and 16%, P<.05, respectively) LDL from subjects with normal LDL-C concentrations.

View this table: [in this window] [in a new window]   Table 1. Age-Adjusted Plasma Lipoprotein Concentrations and Receptor-Binding Affinity in Normocholesterolemic (LDL<160 mg/dL) Subjects by Predominant LDL Size Groups

View this table: [in this window] [in a new window]   Table 2. Age-Adjusted Plasma Lipoprotein Concentrations and Receptor-Binding Affinity in Hypercholesterolemic (LDL160 mg/dL) Subjects by Predominant LDL Size Groups

In both groups (low– and high–LDL-C; Tables 1 and 2) TG concentrations were significantly (P=.0001) increased and HDL-C concentrations significantly (P=.001) decreased with decreasing LDL size. Mean LDL-C concentrations were similar among subjects in the three LDL size categories in both the normal– and high–LDL-C groups. Binding affinity was significantly and positively associated with age (r=.35, P=.009) in subjects with normal LDL-C concentrations. Therefore, we included age as a covariate in our analysis. As shown in Tables 1 and 2, LDL size groups (P=.05) and age (P=.006) were significantly associated with binding affinity in subjects with normal but not in those with high LDL-C concentrations.

Study II: LDLR Binding Affinity of LDL Subfractions From Normolipidemic Subjects With Patterns A and B
The mean LDL peak particle diameter and mean percent area distribution for the three LDL subfractions from subjects with patterns A and B are shown in Table 3. Predominant peak particle diameters and relative size distributions for the three subfractions were similar in pattern A and pattern B subjects. The mean predominant peak particle diameters in pattern A and pattern B subjects were 271 and 274 Å for LDL-I, 264 and 262 Å for LDL-II, and 254 and 250 Å for LDL III, respectively. The relative mass of subfraction LDL-I as determined by protein-stained GGE gels was predominantly in the large-LDL size range (A=77% and B=75%). For subfraction LDL-II 50% of the mass was in the medium-LDL size range (A=51% and B=43%) and most of the remainder was in the large-LDL size range (A=32% and B=30%). For subfraction LDL-III most of the mass was in the small-LDL size range (A=62% and B=85%).

View this table: [in this window] [in a new window]   Table 3. Relative Size Distribution and Peak Particle Diameters in Three LDL Subfractions From Pattern A and Pattern B Subjects

Table 4 shows K_d and B_max values for the three LDL subfractions in all subjects. In the group as a whole (n=6 values), the binding affinity of LDL-III was reduced by 30% (ie, higher K_d, P.05) and B_max was reduced by 25% (P.01) compared with LDL-II. There were no differences in K_d and B_max values between LDL-II and LDL-I. In pattern A subjects, LDL-II had the highest binding affinity (9.2±1.4 µg/mg cell protein). Compared with that for LDL-II, the binding affinities for LDL-I and LDL-III were 37% lower and 36% lower, respectively. Furthermore, B_max was significantly (P.05) reduced by 17% in subfraction LDL-III compared with that in LDL-II. Our finding of a reduced binding affinity for LDL-I and LDL-III relative to LDL-II in these pattern A subjects is similar to the observations in Study I for unfractionated LDL preparations. In contrast, for pattern B subjects, we found that the binding affinity of LDL-I was higher than those for LDL-II (15% lower K_d) and LDL-III (32% lower K_d). Furthermore, B_max was significantly (P.01) lower (by 32%) in LDL-III than in LDL-II. When the results for pattern A subjects were compared with those for pattern B, LDL-I in the latter group had significantly (P=.05) higher binding affinity (37% lower K_d) than did LDL-I from pattern A subjects, but there were no significant differences between pattern A and pattern B subjects for the binding affinity of LDL-II and LDL-III or in B_max values for any fraction.

View this table: [in this window] [in a new window]   Table 4. Receptor-Binding Affinity of 125I-Labeled LDL Subfractions to Fibroblasts at 4°C and ApoE:ApoB Molar Ratios for Pattern A and B Subjects

On the basis of these results we wished to examine whether the differences in LDL-I binding affinity between pattern A and pattern B subjects were related to different contents of apoE, which has higher affinity for the LDLR than does apoB.²⁷ As shown in Table 4 we found that LDL-I from pattern B subjects had a significantly (P=.001) higher apoE to apoB molar ratio than did LDL-I from pattern A subjects. Assuming that there is 1 apoB molecule per LDL particle, we estimated that on average there is 1 apoE per 5 LDL particles in pattern B LDL-I but 1 per 18 for pattern A LDL-I (P=.001). The apoE content of pattern B LDL-I was also significantly higher than those for the other two pattern B subfractions and for LDL-II in pattern A.

The Figure shows the effects of blocking apoE-mediated binding of LDL-I particles with monoclonal antibody ID7. The binding affinity of LDL-I from pattern B subjects was inhibited 1.4-fold in the presence of ID7 (panel B), whereas binding of LDL-I from pattern A subjects was unaffected (panel A). With 1D7 mean binding affinity for the two pattern B LDL-I preparations was 43% lower (K_d=20.0 and 19.2 µg/mg cell protein, respectively) than that for pattern A LDL-I (13.2 and 14.2 µg/mg cell protein, respectively).

View larger version (19K): [in this window] [in a new window]   Figure 1. Scatchard plots of LDLR binding of 125I-labeled LDL-I subfractions from pattern A and pattern B subjects, incubated at 4°C in the presence or absence of monoclonal antibody 1D7, which specifically inhibits apoE-mediated binding. Cells were grown to confluence in 35-mm plastic wells by incubation with LPDS for 48 hours to upregulate the receptors prior to assay. Media containing 0.125-12 mg protein per milliliter of 125I-labeled LDL-I (d=1.026-1.32 g/mL) from 2 pattern A and 2 pattern B subjects were incubated with human fibroblasts in duplicate wells for 5 hours at 4°C in the presence or absence of a 10-fold excess (by protein mass) of 1D7. Nonspecific binding was determined by incubation with a 100-fold excess of unlabeled LDL-II (d=1.020-1.050 g/mL; diameter=262 Å). For the 2 pattern A subjects, LDL-I with no antibody had a Kd=13.7 and 13.2 µg/mg cell protein; with 1D7 antibody, Kd=13.2 and 14.2 µg/mg cell protein. For the 2 pattern B subjects, LDL-I with no antibody had a Kd=8.5 and 7.8 mg/mL cell protein; with 1D7 antibody, Kd=20.0 and 19.2 mg/mL cell protein.

Study III: LDLR Function in Skin-Derived Fibroblast Cultures From Subjects With LDL Subclass Patterns A and B
As shown in Table 5, we found no significant differences in K_d values for ¹²⁵I-labeled LDL or ¹²⁵I-labeled apoE HDL_c at 4°C between fibroblasts from pattern A versus those from pattern B subjects. B_max values were also similar for the two groups. Thus, on the basis of direct binding assays there is no evidence of altered binding of apoB or apoE to the LDLR of pattern B subjects.

View this table: [in this window] [in a new window]   Table 5. Receptor-Binding Affinity of 125I-Labeled LDL and 125I-Labeled ApoE-HDLC to Fibroblasts at 4°C Derived From Pattern A vs Pattern B Subjects

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Several previous studies^{14 15 16 17 18} have reported alterations in the receptor-binding affinity of LDL particles of differing size and density. However, there is incomplete information on the relative binding affinity of unfractionated LDL preparations in subjects with different predominant LDL peak particle sizes. We found that LDL from normolipidemic subjects with a predominance of medium-size LDL exhibited stronger LDLR binding affinity than did the LDL from subjects with a predominance of large or small LDL. Based on the correspondence between peak LDL size and density,¹ these findings in unfractionated LDL preparations are consistent with previous observations in isolated density subfractions from normolipidemic subjects¹⁷ and with the present results, suggesting that in normolipidemia the binding characteristics of LDL preparations are mostly influenced by the binding characteristics of the most abundant LDL species. Decreased receptor-binding affinity of large, buoyant LDL particles may be due to steric hindrance associated with crowding of LDL particles on receptor lattices,¹⁸ whereas decreased receptor-binding affinity of small, dense LDL may be due to changes in apoB conformation that influence the interactions of apoB with the receptor.³³ It has been reported that decreased binding affinity of small LDL is not due to differences in the neutral lipid of the LDL core, because reductions in receptor-binding affinity of smaller, denser LDL from hypertriglyceridemic subjects is independent of TG concentrations.³⁴ Furthermore, it has also been shown that TG enrichment of LDL does not appear to affect the conformation of apoB in the receptor-binding domain.³⁵

The weaker binding affinities of large, buoyant and small, dense LDL compared with LDL of medium size and density may have complex metabolic consequences that affect the atherogenicity of these particles. Decreased binding may be associated with increased plasma residence time and therefore possibly greater opportunity for potentially atherogenic intravascular and cellular modifications. On the other hand, lower receptor affinity may lead to receptor upregulation, although this phenomenon was not reflected in lower concentrations of plasma LDL-C in the subject subgroups with reduced receptor affinity in the present report.

In contrast to these observations in normocholesterolemic subjects, LDLR binding affinity was not reduced in those subjects with large and small LDL who had elevated LDL-C levels (160 mg/dL). Overall, LDL binding affinity was significantly greater in subjects with a high compared with those with a normal LDL-C concentration. Our data also showed a significant association between increasing age and higher affinity for the LDLR. It has been reported that age is associated with increased LDL-C concentrations,³⁶ smaller LDL particles,³⁷ and decreased LDL fractional catabolic rates.³⁸ Furthermore, data from animal studies have shown that older age is associated with reduced expression of LDLRs on liver cell membranes.³⁹

It is known that LDL particles are heterogeneous with respect to their affinity for LDLR- versus non-LDLR–mediated uptake.⁴⁰ Thus, with LDLR activity suppressed, particles with higher receptor affinity that are primarily dependent on receptor function for normal LDL clearance may accumulate in plasma to a greater extent than would LDL with reduced receptor affinity, which might be capable of clearance through non-LDLR–mediated pathways. Resulting enrichment of subfractions I and III with these particles would increase the overall binding affinity. Alternatively, downregulation of LDLRs could alter LDL metabolism so that changes in LDL composition and structure that create high affinity for the LDLR could occur. For example, such particles in hypercholesterolemic subjects could contain higher amounts of apoE, or there could be increased expression of the receptor recognition sites of apoB as a result of intravascular remodeling.

A major objective of our study was to determine whether differences in receptor-binding affinity of LDL I, II, and III from subjects with pattern A differed from those for equivalent fractions from pattern B subjects. Although reductions in receptor-binding affinity in LDL-III relative to LDL-II were similar in pattern A and B subjects, the binding affinity of LDL-I was significantly greater for pattern B. On the basis of the higher apoE content in LDL-I fractions in pattern B subjects and the substantial reduction in receptor affinity resulting from incubation with a monoclonal antibody specific for the receptor binding-domain of apoE, we conclude that greater amounts of apoE-containing particles are responsible for this result. However, we cannot rule out the possibility that the experimental conditions in our studies were not optimal for eliminating the contribution of apoE to LDL-I binding. If one assumes that apoE-mediated LDLR binding in pattern A subjects is blocked by incubation with the antibody, then the results indicate reduced apoB-mediated receptor binding of LDL-I, relative to LDL-II, from both pattern A and pattern B subjects (consistent with earlier results¹⁷ and our present findings for unfractionated LDL from normocholesterolemic subjects with large LDL). The residual binding affinity of LDL-I from pattern B subjects appeared much weaker than that from pattern A subjects. This result must be interpreted cautiously, because the number of study subjects was small and the possibility exists that binding of the monoclonal antibody may have sterically interfered with apoB-mediated binding. This possibility is currently being explored by studies of apoE-free LDL preparations that have been isolated by anti-apoE immunoaffinity chromatography.

The reason for the greater apoE content in LDL-I from pattern B subjects is unknown. IDL levels are known to be higher in subjects with small LDL,^{4 7} and it is possible that the size and density distributions of these apoE-containing particles overlap those for LDL-I in pattern B subjects. However, we cannot rule out the possibility that ultracentrifugal redistribution of IDL or apoE is responsible for these results. Preliminary experiments with chromatographically separated lipoproteins (P.J. Blanche et al, unpublished data, 1995) indicate that levels of apoE-containing particles in the size range of small IDL and large LDL are indeed higher for pattern B. The reason for this finding is unknown, but Table 5 of the present report rules out the possibility of a defect in apoE- (as well as apoB-) binding properties of fibroblast LDLRs in pattern B subjects.

Previous studies have suggested linkage of the gene for pattern B to a locus on chromosome 19 near the LDLR gene.¹² However, according to the direct binding assays in this study, we found no evidence for altered binding of apoB or apoE to the LDLR of pattern B subjects, consistent with recent evidence (J.K. Naggert et al, unpublished data, 1995) of the absence of functional mutations in the structural position of the LDLR gene in pattern B subjects. Thus, metabolic abnormalities in pattern B subjects that may be responsible for the higher apoE content of large, buoyant LDL include increased production of apoE-containing, TG-rich lipoproteins and/or greater plasma retention of these particles due to impaired lipolysis or clearance. It is possible that, in turn, accumulation of these particles contributes to the higher CAD risk in pattern B subjects.^{8 41}

Taken together, our data confirm that LDL subspecies are heterogeneous in their affinity for the LDLR. In normocholesterolemic but not hypercholesterolemic subjects, both large LDL-I and small LDL-III particles show reduced binding affinity. Increased receptor-binding affinity is associated with age and with increased LDL-C concentrations. Variations in apoE content and differences in particle size that may affect apoB conformation and exposure of its receptor-binding domains contribute to the differences in LDLR binding among LDL subclasses and individuals and may result in differing metabolic behavior of these particles in vivo.

	Selected Abbreviations and Acronyms

 

CAD = coronary artery disease DMEM = Dulbecco's modified Eagle's medium GGE = gradient gel electrophoresis HDLc = cholesterolemic HDL HDL-C = HDL cholesterol LDL-C = LDL cholesterol LDLR = LDL receptor LPDS = lipoprotein-deficient serum TG(s) = triglyceride(s)

	Acknowledgments

 
This work was supported by the National Institutes of Health program project grant HL-18574 from the National Heart, Lung, and Blood Institute and a grant from the National Dairy Promotion and Research Board, administered in cooperation with the National Dairy Council, and was conducted at the Ernest Orlando Lawrence Berkeley National Laboratory through the US Department of Energy under contract No. DE-AC03-76SF00098 and at the Gladstone Institute of Cardiovascular Disease, San Francisco, California. This work was done during the tenure of a Research Fellowship from the American Heart Association, California Affiliate (Dr Campos). We thank Dr Thomas Bersot at the Gladstone Institute of Cardiovascular Disease for carrying out the human skin punch biopsies; Patricia Blanche and the staff of Donner Lipoprotein Core Laboratory for performing the lipid, lipoprotein, and apolipoprotein measurements; and Adelle Cavanaugh for assistance in carrying out the clinical protocols.

Received August 25, 1995; accepted December 20, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Krauss RM, Burke DJ. Identification of multiple subclasses of plasma low density lipoproteins in normal humans. J Lipid Res. 1982;23:97-104. [Abstract]

2. Shen MMS, Krauss RM, Lindgren FT, Forte TM. Heterogeneity of serum low density lipoproteins in normal human subjects. J Lipid Res. 1981;22:236-244. [Abstract]

3. Campos H, Blijlevens E, McNamara JR, Ordovas JM, Posner BM, Wilson PWF, Castelli WP, Schaefer EJ. Low density lipoprotein particle size distribution: results from the Framingham Offspring Study. Arterioscler Thromb. 1992;12:1410-1419. [Abstract/Free Full Text]

4. Krauss RM. Relationship of intermediate and low-density lipoprotein subspecies to risk of coronary artery disease. Am Heart J. 1987;113:578-582. [Medline] [Order article via Infotrieve]

5. Krauss RM. The tangled web of coronary risk factors. Am J Med. 1991;90(suppl 2A):36s-41s.

6. Austin MA, King M-C, Vranizan KM, Newman B, Krauss RM. Inheritance of low-density lipoprotein subclass patterns: results of complex segregation analysis. Am J Hum Genet. 1988;43:838-846. [Medline] [Order article via Infotrieve]

7. Austin MA, King M-C, Vranizan KM, Krauss RM. Atherogenic lipoprotein phenotype: a proposed genetic marker of coronary heart disease risk. Circulation. 1990;82:495-506. [Abstract/Free Full Text]

8. Austin MA, Breslow JL, Hennekens CH, Buring JE, Willett WC, Krauss RM. Low-density lipoprotein subclass patterns and risk of myocardial infarction. JAMA. 1988;260:1917-1921. [Abstract/Free Full Text]

9. Campos H, Genest J, Blijlevens E, McNamara JR, Jenner JL, Ordovas JM, Wilson PWF, Schaefer EJ. Low density lipoprotein particle size and coronary artery disease. Arterioscler Thromb. 1992;12:187-195. [Abstract/Free Full Text]

10. Krauss RM. Low-density lipoprotein subclasses and risk of coronary artery disease. Curr Opin Lipidol. 1991;2:248-252.

11. Austin MA, Brunzell JD, Fitch WL, Krauss RM. Inheritance of low density lipoprotein subclass patterns in familial combined hyperlipidemia. Arteriosclerosis. 1990;10:520-530. [Abstract/Free Full Text]

12. Nishina PM, Johnson JP, Naggert JK, Krauss RM. Linkage of atherogenic lipoprotein phenotype to the low density lipoprotein receptor locus on the short arm of chromosome 19. Proc Natl Acad Sci U S A. 1992;89:708-712. [Abstract/Free Full Text]

13. Rotter JI, Bu X, Cantor RM, Warden CH, Brown J, Gray RJ, Blanche PJ, Krauss RM, Lusis AJ. Multiple genes determine LDL particle size phenotype in coronary artery disease families. Circulation. 1994;90(pt 2):I-509. Abstract.

14. Swinkels DW, Demacker PNM, Hak-Lemmers HLM, Mol MJTM, Yap SH, van't Laar A. Some metabolic characteristics of low-density lipoprotein subfractions, LDL-1 and LDL-2: in vitro and in vivo studies. Biochim Biophys Acta. 1988;960:1-9. [Medline] [Order article via Infotrieve]

15. Jaakkola O, Solakivi T, Ylä-Herttuala S, Nikkari T. Receptor-mediated binding and degradation of subfractions of human plasma low-density lipoprotein by cultured fibroblasts. Biochim Biophys Acta. 1989;1005:118-122. [Medline] [Order article via Infotrieve]

16. Swinkels DW, Hendriks JCM, Demacker PNM, Stalenhoef AFH. Differences in metabolism of three low density lipoprotein subfractions in Hep G2 Cells. Biochim Biophys Acta. 1990;1047:212-222. [Medline] [Order article via Infotrieve]

17. Nigon F, Lesnik P, Rouis M, Chapman MJ. Discrete subspecies of human low density lipoproteins are heterogeneous in their interaction with cellular LDL receptor. J Lipid Res. 1991;32:1741-1753. [Abstract]

18. Chappell DA, Fry GL, Waknitz MA, Berns JJ. Ligand size as a determinant of catabolism by the low density lipoprotein (LDL) receptor pathway. J Biol Chem. 1991;266:19296-19302. [Abstract/Free Full Text]

19. The Expert Panel. Report of the National Cholesterol Education Program expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. Arch Intern Med. 1988;148:36-39. [Abstract/Free Full Text]

20. Nichols AV, Krauss RM, Musliner TA. Nondenaturing polyacrylamide gradient gel electrophoresis. Methods Enzymol. 1986;128:417-431. [Medline] [Order article via Infotrieve]

21. Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of low density lipoproteins in cultured cells. Methods Enzymol. 1983;98:241-260. [Medline] [Order article via Infotrieve]

22. Innerarity TL, Pitas RE, Mahley RW. Lipoprotein-receptor interactions. Methods Enzymol. 1986;129:542-566. [Medline] [Order article via Infotrieve]

23. Arnold KS, Innerarity RL, Pitas RE, Mahley RW. Lipoprotein-receptor interactions. In: Converse CA, Skinner ER, eds. Lipoprotein Analysis: A Practical Approach. Oxford, England: IRL Press; 1991:145-168.

24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. [Free Full Text]

25. Milne RW, Douste-Blazy PH, Retegui L, Marcel YL. Characterization of monoclonal antibody against human apolipoprotein E. J Clin Invest. 1981;68:111-117.

26. Dreon DM, Fernstrom HA, Miller B, Krauss RM. Low-density lipoprotein subclass patterns and response to a reduced-fat diet in men. FASEB J. 1994;8:121-126. [Abstract]

27. Pitas RE, Innerarity TL, Arnold KS, Mahley RW. Rate and equilibrium constants for binding of apo-E HDL_C (a cholesterol-induced lipoprotein) and low density lipoproteins to human fibroblasts: evidence of multiple receptor binding of apo-E HDL_C. Proc Natl Acad Sci U S A. 1979;76:2311-2315. [Abstract/Free Full Text]

28. Warnick GR, Nguyen T, Albers JJ. Comparison of improved precipitation methods for quantification of high density lipoprotein cholesterol. Clin Chem. 1985;31:217-222. [Abstract/Free Full Text]

29. Mancini G, Carbonara AO, Heremans JF. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry. 1965;2:235-254. [Medline] [Order article via Infotrieve]

30. Tijssen P. Practice and theory of enzyme immunoassays. In: Burdon RH, van Knippenberg PH, eds. Laboratory Techniques in Biochemistry and Molecular Biology, Volume 15. New York, NY: Elsevier; 1985:337-343.

31. Kahlon TS, Shore VG, Lindgren FT. Heterogeneity of molecular weight and apolipoproteins in low density lipoproteins of healthy human males. Lipids. 1992;27:1055-1057. [Medline] [Order article via Infotrieve]

32. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science. 1988;240:622-630. [Abstract/Free Full Text]

33. Teng B, Sniderman A, Krauss RM, Kwiterovich RO Jr, Milne RW, Marcel IL. Modulation of apolipoprotein B antigenic determinants in human low density lipoprotein subclasses. J Biol Chem. 1985;260:5067-5072. [Abstract/Free Full Text]

34. Galeano NF, Milne R, Marcel YL, Walsh MT, Levy E, Ngu'yen TD, Gleeson A, Arad Y, Witte L, Al-Haideri M, Rumsey SC, Deckelbaum RJ. Apoprotein B structure and receptor recognition of TG-rich low density lipoprotein (LDL) is modified in small LDL but not in triglyceride-rich LDL of normal size. J Biol Chem. 1993;269:511-519. [Abstract/Free Full Text]

35. McKeone BJ, Patsch JR, Pownall HJ. Plasma triglyceride determines low density lipoprotein composition, physical properties, and cell-specific binding in cultured cells. J Clin Invest. 1993;91:1926-1933.

36. Heiss G, Tamir I, Davis CE, Tyroler HA, Rifkind BM, Schonfeld G, Jacobs D, Frantz ID. Lipoprotein-cholesterol distributions in selected North American populations: the Lipid Research Clinics Program Prevalence Study. Circulation. 1980;61:302-315. [Free Full Text]

37. McNamara JR, Campos H, Ordovas JM, Wilson PWF, Schaefer EJ. Effect of gender and lipid status on low density lipoprotein subfraction distribution: results from the Framingham Offspring Study. Arteriosclerosis. 1987;7:483-490. [Abstract/Free Full Text]

38. Ericsson S, Eriksson M, Vitols S, Einarsson K, Berglund L, Angelin B. Influence of age on the metabolism of plasma low density lipoproteins in healthy males. J Clin Invest. 1991;87:591-596.

39. Mahley RW, Hui DY, Innerarity TL, Weisgraber KH. Two independent lipoprotein receptors on hepatic membranes of the dog, swine, and man: apo-B,E and apo-E receptors. J Clin Invest. 1981;68:1197-1206.

40. Rumsey SC, Obunike JC, Arad Y, Deckelbaum RJ, Goldberg IJ. Lipoprotein lipase-mediated uptake and degradation of low density lipoproteins by fibroblasts and macrophages. J Clin Invest. 1992;90:1504-1512.

41. Krauss RM, Lindgren FT, Williams PT, Kelsey SF, Brensike J, Vranizan K, et al. Intermediate-density lipoproteins and progression of coronary artery disease in hypercholesterolemic men. Lancet. 1987;2:62-66.[Medline] [Order article via Infotrieve]

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