Characterization of human apolipoprotein B100 oligosaccharides in LDL subfractions derived from normal and hyperlipidemic plasma: deficiency of alpha-N-acetylneuraminyllactosyl-ceramide in light and small dense LDL particles

The carbohydrate composition of apolipoprotein (apo) B100, particularly its degree of sialylation, may contribute to the atherogenic properties of low-density lipoprotein (LDL). We analyzed LDL apoB100 glycans derived from normolipidemic, hypercholesterolemic, and hypertriglyceridemic diabetic subjects. Using exoglycosidase carbohydrate sequencing and matrix-assisted laser desorption/ionization mass spectrometry to analyze fluorescently labeled oligosaccharides, we report evidence for several carbohydrates not previously identified on apoB100, including truncated complex biantennary N-glycans and hybrid N-glycans. The distribution and diversity of the apoB100 glycans isolated from all individuals was highly conserved. The N-glycan composition of apoB100 derived from five LDL subpopulations (LDL1, d = 1.018-1.023; LDL2, d = 1.023-1.030; LDL3, d = 1.030-1.040; LDL4, d = 1.040-1.051; LDL5, d = 1.051-1.065 g/ml) did not vary in normolipidemic or hypercholesterolemic subjects. Furthermore, we found no evidence for "desialylated" apoB100 glycans in any of the samples analyzed. Analysis of the most abundant LDL ganglioside, alpha-N-acetylneuraminyllactosyl-ceramide, revealed a deficiency in small dense LDL and in the most buoyant subpopulation. These data provide a novel explanation for the apparent deficiency of sialic acid in small dense LDL and indicate that the global apoB100 N-glycan composition is invariable in the patient groups studied.     

A novel and simple method for quantification of small,dense LDL

A preponderance of small, dense (sd) LDL is strongly associated with the development of coronary heart disease, but the method for the measurement of sd LDL is too laborious for clinical use. We report a simple method for the quantification of sd LDL that is applicable to an autoanalyzer. This method consists of two steps: first, to precipitate the lipoprotein of density (d) <1.044 g/ml using heparin-magnesium; and second, to measure LDL-cholesterol in the supernatant by the homogeneous method or apolipoprotein B (apoB) by an immunoturbidometric assay. The cholesterol and apoB values obtained by the precipitation method (45 +/- 26 and 33 +/- 20 mg/dl, respectively) were similar to those obtained in the lipoprotein (d = 1.044-1.063) separated by ultracentrifugation (42 +/- 22 and 31 +/- 17 mg/dl, respectively), and there was an excellent correlation between the two methods for sd LDL-cholesterol (y = 1.05X + 1, r = 0.88, n = 69) and apoB (y = 1.07X, r = 0.90). Sd LDL values had a significant inverse correlation with LDL size. A high correlation was found between sd LDL-cholesterol and apoB values (r = 0.94). Sd LDL value was related to triglyceride, apoB, and LDL-cholesterol, but not to the buoyant LDL level. These results suggest that this precipitation method is a simple and rapid method for the measurement of sd LDL concentration.     

Acute impact of apheresis on oxidized phospholipids in patients with familial hypercholesterolemia

We measured oxidized phospholipids (OxPL), lipoprotein (a) [Lp(a)], and lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) pre- and postapheresis in 18 patients with familial hypercholesterolemia (FH) and with low(～10 mg/dl; range 10-11 mg/dl), intermediate (～50 mg/dl; range 30-61 mg/dl), or high (>100 mg/dl; range 78-128 mg/dl) Lp(a) levels. By using enzymatic and immunoassays, the content of OxPL and Lp-PLA(2) mass and activity were quantitated in lipoprotein density fractions plated in microtiter wells, as well as directly on apoB-100, Lp(a), and apoA-I immunocaptured within each fraction (i.e., OxPL/apoB and Lp-PLA(2)/apoB). In whole fractions, OxPL was primarily detected in the Lp(a)-containing fractions, whereas Lp-PLA(2) was primarily detected in the small, dense LDL and light Lp(a) range. In lipoprotein capture assays, OxPL/apoB and OxPL/apo(a) increased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apoB and Lp-PLA(2)/apoA-I levels were highest in the low Lp(a) group but decreased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apo(a) was lowest in patients with low Lp(a) levels and increased proportionally with increasing Lp(a) levels. Apheresis significantly reduced levels of OxPL and Lp-PLA(2) on apoB and Lp(a) (50-75%), particularly in patients with intermediate and high Lp(a) levels. In contrast, apheresis increased Lp-PLA(2)-specific activity (activity/mass ratio) in buoyant LDL fractions. The impact of apheresis on Lp(a), OxPL, and Lp-PLA(2) provides insights into its therapeutic benefits beyond lowering apoB-containing lipoproteins.     

Influence of LDL apheresis on LDL subtypes in patients with coronary heart disease and severe hyperlipoproteinemia

Epidemiologic studies and in vitro experiments indicate that low density lipoprotein (LDL) subtypes differ concerning their atherogenic potential. Small, dense LDL are more atherogenic than large, buoyant LDL. LDL apheresis is a potent therapeutic modality to lower elevated LDL-cholesterol. It is unknown whether such therapy induces a shift in the LDL subtype distribution. In this study we evaluated the influence of LDL apheresis on the LDL subtype distribution in patients with CHD and familial hypercholesterolemia (FH, n = 22), combined hyperlipidemia (CHLP, n = 6), or Lp[a]-hyperlipoproteinemia (Lp[a]-HLP, n = 4) regularly treated by LDL apheresis (immunoadsorption (n = 14), HELP apheresis (n = 8), dextran sulfate adsorption (n = 7), cascade filtration (n = 3)). On the basis of 6 LDL subfractions (d 1.020;-1.057 g/mL) isolated by density gradient ultracentrifugation the LDL-density profile was determined in each patient before and after apheresis. There was a relative increase of LDL-subfractions 1, 2, and 3 (P < 0.01, P < 0. 05, and P < 0.01, respectively) and a concomitant decrease of LDL subfractions 5 and 6 (P < 0.05) after apheresis. Subgroup analysis indicates that the degree of the small, dense LDL reduction was much more prominent in patients with CHLP compared to patients with FH or Lp[a]-HLP, whereas the type of apheresis technique had no effect. The extent of small, dense LDL reduction correlated with the preapheresis concentrations of small, dense LDL and triglycerides but not with the extent of triglyceride reduction.We conclude that LDL apheresis not only decreases LDL mass, but also improves LDL-density profile, particularly in patients with CHLP.     

Apolipoprotein B metabolism and the distribution of VLDL and LDL subfractions

Apolipoprotein B (apoB) metabolism was investigated in 20 men with plasma triglyceride 0.66-2.40 mmol/l and plasma cholesterol 3.95-6. 95 mmol/l. Kinetics of VLDL(1) (S(f) 60-400), VLDL(2) (S(f) 20-60), IDL (S(f) 12-20), and LDL (S(f) 0;-12) apoB were analyzed using a trideuterated leucine tracer and a multicompartmental model which allowed input into each fraction. VLDL(1) apoB production varied widely (from 5.4 to 26.6 mg/kg/d) as did VLDL(2) apoB production (from 0.18 to 8.4 mg/kg/d) but the two were not correlated. IDL plus LDL apoB direct production accounted for up to half of total apoB production and was inversely related to plasma triglyceride (r = -0.54, P = 0.009). Percent of direct apoB production into the IDL/LDL density range (r = 0.50, P < 0.02) was positively related to the LDL apoB fractional catabolic rate (FCR). Plasma triglyceride in these subjects was determined principally by VLDL(1) and VLDL(2) apoB fractional transfer rates (FTR), i.e., lipolysis. IDL apoB concentration was regulated mainly by the IDL to LDL FTR (r = -0.71, P < 0.0001). LDL apoB concentration correlated with VLDL(2) apoB production (r = 0.48, P = 0.018) and the LDL FCR (r = -0.77, P < 0. 001) but not with VLDL(1), IDL, or LDL apoB production. Subjects with predominantly small, dense LDL (pattern B) had lower VLDL(1) and VLDL(2) apoB FTRs, higher VLDL(2) apoB production, and a lower LDL apoB FCR than those with large LDL (pattern A). Thus, the metabolic conditions that favored appearance of small, dense LDL were diminished lipolysis of VLDL, resulting in a raised plasma triglyceride above the putative threshold of 1.5 mmol/l, and a prolonged residence time for LDL. This latter condition presumably permitted sufficient time for the processes of lipid exchange and lipolysis to generate small LDL particles.     

Measurment of rhesus monkey (Macaca mulatta) apolipoprotein B in serum by radioimmunoassay: comparison of immunoreactivities of rhesus and human low density lipoproteins.

A sensitive and specific double antibody radio-immunoassay for the major apolipoprotein (apoB) of rhesus (Macaca mulatta) serum very low density lipoprotein (VLDL) and low density lipoprotein (LDL) is described. The anti-serum was raised to LDL (d 1.030-1.040 g/ml) and the LDL(2) (d 1.020-1.050 g/ml) was labeled with (125)I by the chloramine-T or iodine monochloride method. The assay, which was sensitive to 0.02-0.5 micro g of LDL(2), had an inter-assay coefficient of variation of 4.5%. This assay was successfully used to measure apoB in the whole serum and low density lipoproteins of control monkeys maintained on a standard Purina monkey chow (PMC) diet and of three groups of monkeys fed atherogenic diets: an "average American diet," a 25% peanut oil and 2% cholesterol-supplemented PMC diet, and a 25% coconut oil and 2% cholesterol-supplemented PMC diet. The control monkeys (n = 13) had a serum cholesterol of 146 +/- 28 mg/dl and an apoB of 50 +/- 18 mg/dl. In the monkeys maintained on the atherogenic diets the serum apoB was elevated: 103 +/- 28 mg/dl (American), 102 +/- 35 mg/dl (peanut oil), and 312 +/- 88 mg/dl (coconut oil). The values for serum total cholesterol were 333 +/- 65 mg/dl (American), 606 +/- 212 mg/dl (peanut oil), and 864 +/- 233 mg/dl (coconut oil) and were elevated relative to controls (P < 0.001). For each of the diets, total serum cholesterol correlated with serum apoB (P < 0.001). The slopes of the regression lines of serum apoB vs. cholesterol for the monkeys on the PMC, American, and coconut oil diets were similar (m = 0.531, 0.401, and 0.359, respectively), but differed from that of monkeys on the peanut oil diet (m = 0.121). The immunoreactivities of rhesus and human LDL were compared using specific antisera raised against these antigens. In homologous assay systems, monkey and human LDL exhibited unique immunological determinants. The same results were obtained with the delipidated preparations of the two LDLs using antisera raised against either monkey or human apoB. Crossover studies using a heterologous tracer with each anti-serum resulted in the selection of a specific population of antibodies directed against antigenic sites shared by these two LDL species.     

Acute effects of low density lipoprotein apheresis on metabolic parameters of apolipoprotein B

Apheresis is a treatment option for patients with severe hypercholesterolemia and coronary artery disease. It is unknown whether such therapy changes kinetic parameters of lipoprotein metabolism, such as apolipoprotein B (apoB) secretion rates, conversion rates, and fractional catabolic rates (FCR). We studied the acute effect of apheresis on metabolic parameters of apoB in five patients with drug-resistant hyperlipoproteinemia, using endogenous labeling with D(3)-leucine, mass spectrometry, and multicompartmental modeling. Patients were studied prior to and immediately after apheresis therapy. The two tracer studies were modeled simultaneously, taking into account the non-steady-state concentrations of apoB. The low density lipoprotein (LDL)-apoB concentration was 120+/-32 mg dl(-1) prior to and 52+/-18 mg dl(-1) immediately after apheresis therapy. The metabolic studies indicate that no change in apoB secretion (13.9+/- 4.9 mg kg(-1) day(-1)) is required to fit the tracer and apoB mass data obtained before and after apheresis and that in four of the five patients the LDL-apoB FCR (0.21+/-0.02 day(-1)) was not altered after apheresis. In one subject the LDL-apoB FCR temporarily increased from 0.22 day(-1) to 0.35 day(-1) after apheresis. The conversion rate of very low density lipoprotein (VLDL)-apoB to LDL-apoB is temporarily decreased from 76 to 51% after apheresis and thus less LDL-apoB is produced after apheresis. We conclude that an acute reduction of LDL-apoB concentration does not affect apoB secretion or LDL-apoB FCR, but that apoB conversion to LDL is temporarily decreased. Thus, in most patients the decreased rate of delivery of neutral lipids or apoB to the liver does not result in an upregulation of LDL receptors or in decreased apoB secretion.     

Density distribution of electronegative LDL in normolipemic and hyperlipemic subjects

The density distribution of electronegative LDL [LDL(-)], a cytotoxic and inflammatory fraction of LDL present in plasma, was studied in 10 normolipemic (NL), 6 FH, and 11 hypertriglyceridemic (HTG) subjects. Six LDL subclasses of increased density (LDL1 to LDL6) were isolated by density-gradient ultracentrifugation (DGU). NL and FH subjects showed prevalence of light LDL, whereas HTG subjects showed prevalence of dense LDL. LDL(-) proportion was determined from total LDL or LDL-density subclasses by anion-exchange chromatography. LDL from FH patients had increased LDL(-) (35.1 +/- 9.9%) compared with LDL from NL and HTG subjects (9.4 +/- 2.3% and 12.3 +/- 4.3%, respectively). Most LDL(-) was contained in dense subclasses in NL (LDL4-6, 67.7 +/- 3.1%) whereas most of LDL(-) from FH patients were contained in light LDL subclasses (LDL1-3) (86.2 +/- 1.6%). In these subjects, simvastatin therapy decreased LDL(-) to 28.2 +/- 6.7% and 21.2 +/- 5.6% at 3 and 6 months of treatment, respectively, due mainly to decreases in light LDL subclasses. In HTG subjects, half LDL(-) was contained in dense LDL subclasses (LDL4-6, 46.1 +/- 2.0%). Non-denaturing acrylamide gradient gel electrophoresis concurred with DGU data, as LDL(-) from NL showed a single band of lower size than non-electronegative LDL [LDL(+)], whereas LDL(-) from FH and HTG presented bands of greater size than its respective LDL(+). These results reveal the existence of light and dense LDL(-), indicate that hyperlipemia could promote the formation of light LDL(-) and suggest that LDL(-) could have different origins.     

Impaired binding affinity of electronegative low-density lipoprotein (LDL) to the LDL receptor is related to nonesterified fatty acids and lysophosphatidylcholine content

The binding characteristics of electropositive [LDL(+)] and electronegative LDL [LDL(-)] subfractions to the LDL receptor (LDLr) were studied. Saturation kinetic studies in cultured human fibroblasts demonstrated that LDL(-) from normolipemic (NL) and familial hypercholesterolemic (FH) subjects had lower binding affinity than their respective LDL(+) fractions (P < 0.05), as indicated by higher dissociation constant (K(D)) values. FH-LDL(+) also showed lower binding affinity (P < 0.05) than NL-LDL(+) (K(D), sorted from lower to higher affinity: NL-LDL(-), 33.0 +/- 24.4 nM; FH-LDL(-), 24.4 +/- 7.1 nM; FH-LDL(+), 16.6 +/- 7.0 nM; NL-LDL(+), 10.9 +/- 5.7 nM). These results were confirmed by binding displacement studies. The impaired affinity binding of LDL(-) could be attributed to altered secondary and tertiary structure of apolipoprotein B, but circular dichroism (CD) and tryptophan fluorescence (TrpF) studies revealed no structural differences between LDL(+) and LDL(-). To ascertain the role of increased nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC) content in LDL(-), LDL(+) was enriched in NEFA or hydrolyzed with secretory phospholipase A(2). Modification of LDL gradually decreased the affinity to LDLr in parallel to the increasing content of NEFA and/or LPC. Modified LDLs with a NEFA content similar to that of LDL(-) displayed similar affinity. ApoB structure studies of modified LDLs by CD and TrpF showed no difference compared to LDL(+) or LDL(-). Our results indicate that NEFA loading or phospholipase A(2) lipolysis of LDL leads to changes that affect the affinity of LDL to LDLr with no major effect on apoB structure. Impaired affinity to the LDLr shown by LDL(-) is related to NEFA and/or LPC content rather than to structural differences in apolipoprotein B.     

Concentrations and compositions of plasma lipoprotein subfractions of Lpb5-Lpu1 homozygous and heterozygous swine with hypercholesterolemia

Pigs with two mutant epitopes, Lpb5 of apolipoprotein B (apoB) and Lpu1 of a yet undefined apolipoprotein, specified by a haplotype Lpb5-Lpu1 and fed a cholesterol-free low fat diet show hypercholesterolemia. The purpose of this study was to establish whether a direct relationship exists between the swine lipoprotein concentration/composition and the genotype for the Lpb5-Lpu1 haplotype; i.e., homozygote versus heterozygote. Lipoproteins of fasted plasma from hypercholesterolemic swine, homozygous (HmHC) and heterozygous (HtHC) for Lpb5-Lpu1, and from normolipidemic (NL) pigs of other Lpb-Lpu haplotypes were separated into five layers by density gradient ultracentrifugation. Layer 1 contained particles of d less than 1.019 g/ml and layer 5 particles of d greater than 1.073 g/ml. Layers 2, 3, and 4 represented subfractions of low density lipoproteins (LDL). The plasma total cholesterol (TC) of the HmHC group (300 +/- 84 mg/dl) was different (P less than 0.05) from the HtHC group (200 +/- 80 mg/dl) and in both HmHC and HtHC, TC was significantly higher (P less than 0.0005 and P less than 0.005, respectively) than that of the NL group (69 +/- 14 mg/dl). The elevation in plasma TC was due to the increased TC in layers 2 and 3: a 13- and 7-fold increase in HmHC and a 7- and 4-fold increase in HtHC in layers 2 and 3, respectively. Parallel increases in unesterified cholesterol were observed in these two layers. Marked increases in apoB were also observed in layers 2 and 3 of HmHC and intermediate increases in apoB in the same two layers of HtHC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100

A monoclonal antibody-based direct binding enzyme-linked immunosorbent assay (ELISA) for apoprotein (apo) B-100 has been developed for use as a reference method. The assay uses the two well-characterized monoclonal antibodies, MB24 and MB47. MB47, which recognizes an epitope at the low density lipoprotein (LDL) receptor-binding domain of apoB and is specific for apoB-100, is bound to the microtiter plate as the capture antibody. MB24, which binds an epitope in the amino terminal half of the apoB-100 and identifies both apoB-100 and apoB-48, is conjugated to horseradish peroxidase and is utilized as the indicating antibody. The assay was calibrated with LDL (d 1.030-1.050 g/ml) and the LDL protein was determined by a sodium dodecyl sulfate (SDS) Lowry procedure. The working range of the assay is 0.25-1.25 micrograms/ml. Optimal dilution of whole plasma was found to be 1:2000. In the assay, MB47 bound approximately 97% of the apoB in all low density lipoprotein, and greater than 90% of the apoB in the majority of very low density lipoprotein preparations. Small dense LDL from subjects with familial combined hyperlipidemia (FCHL) and large bouyant LDL from subjects with familial hypercholesterolemia (FH) exhibited binding properties similar to LDL from healthy normolipidemic subjects when tested in the reference ELISA. The intra- and interassay coefficients of variation averaged 2.5% and 6.0%, respectively. Plasma B-100 levels were not influenced by freezing and thawing or storage at 4 degrees C for up to 3 weeks or storage at -70 degrees C for up to 11 months. Excellent agreement was obtained between the reference ELISA and a polyclonal RIA which measures total apoB (r = 0.93, n = 105, mean ELISA B-100 value = 100 mg/dl, mean RIA value = 101 mg/dl, Sy = 9.6). Reference ELISA B-100 values of samples pretreated with bacterial lipase were not significantly increased in most samples with plasma triglyceride levels below 600 mg/dl. To help reduce the large among-laboratories variability of apoB measurements, we recommend that this candidate reference direct binding ELISA be used to assign apoB target values to apoB reference pools.     

Compensatory mechanisms governing the concentration of plasma low density lipoprotein

To evaluate factors regulating the concentrations of plasma low density lipoproteins (LDL), apolipoprotein B metabolism was studied in nine Pima Indians (25 +/- 2 yr, 191 +/- 20% ideal wt) with low LDL cholesterol (77 +/- 7 mg/dl) and apoB (60 +/- 4 mg/dl) and in eight age- and weight-matched Caucasians with similar very low density lipoprotein (VLDL) concentrations, but higher LDL (cholesterol = 104 +/- 18; apoB = 82 +/- 10; P less than 0.05). Subjects received autologous 131I-labeled VLDL and 125I-labeled LDL, and specific activities of VLDL-apoB, intermediate density lipoprotein (IDL)-apoB, and LDL-apoB were analyzed using a multicompartmental model. Synthesis of LDL-apoB was similar (1224 +/- 87 mg/d in Pimas vs 1218 +/- 118 mg/d in Caucasians) but in Pimas the fractional catabolic rate (FCR) for LDL-apoB was higher (0.48 +/- 0.02 vs 0.39 +/- 0.04 d-1, P less than 0.05). In the Pimas, a much higher proportion of VLDL-apoB was catabolized without conversion to LDL (47 +/- 3 vs 30 +/- 5%, P less than 0.01). When all subjects were considered together, LDL-apoB concentrations were negatively correlated with both FCR for LDL-apoB (r = -0.79, P less than 0.0001) and the non-LDL pathway (r = -0.43, P less than 0.05). Also, the direct removal (non-LDL) path was correlated with VLDL-apoB production (r = 0.49, P = 0.03), and the direct removal pathway and FCR for LDL-apoB were correlated (r = 0.49, P = 0.03). In conclusion, plasma LDL appear to be regulated by both the catabolism of LDL and the extent of metabolism of VLDL without conversion to LDL; both of these processes may be mediated by the apoB/E receptor, and appear to increase in response to increasing VLDL production.     

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.     

Conversion of plasma VLDL and IDL precursors into various LDL subpopulations using density gradient ultracentrifugation

The contribution of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) to various low density lipoprotein (LDL) subfractions was examined in three normal subjects and two with familial combined hyperlipidemia. Autologous VLDL + IDL (d less than 1.019 g/ml) or VLDL only (d less than 1.006 g/ml; one subject only) were isolated by sequential ultracentrifugation, iodinated, and injected into each subject. The appearance, distribution, and subsequent disappearance of radioactivity into LDL density subpopulations was characterized using density gradient ultracentrifugation. These techniques help determine the contribution of precursors to various LDL subpopulations defined uniquely for each subject. The results from these studies have suggested: 1) it took up to several days of intravascular processing of precursor-derived LDL before it resembled the distribution of the 'steady-state' plasma LDL protein; 2) plasma VLDL and IDL precursors contributed rapidly to a broad density range of LDL; 3) the radiolabeled plasma precursors did not always contribute to all LDL density subfractions within an individual in proportion to their relative LDL protein mass as determined by density gradient ultracentrifugation; 4) with time, the distribution of the precursor-derived LDL became more buoyant or more dense than distribution of the LDL protein mass; and 5) the kinetic characteristics of precursor-derived particles within LDL changed within a relatively narrow density range and were not always related to the LDL density heterogeneity of each subject. These studies emphasize the complexities of apoB metabolism and the need to design studies to carefully examine the production of various LDL subpopulations, the kinetic fate and interconversions among the subpopulations, and ultimately, their relationship to the development of atherosclerosis.     

Metabolic pathways of apolipoprotein B in heterozygous familial hypercholesterolemia: studies with a [3H]leucine tracer.

The kinetics of apolipoprotein B (apoB) were measured in seven studies in heterozygous, familial hypercholesterolemic subjects (FH) and in five studies in normal subjects, using in vivo tracer kinetic methodology with a [3H]leucine tracer. Very low density (VLDL) and low density lipoproteins (LDL) were isolated ultracentrifugally and LDL was fractionated into high and low molecular weight subspecies. ApoB was isolated, its specific radioactivity was measured, and the kinetic data were analyzed by compartmental modeling using the SAAM computer program. The pathways of apoB metabolism differ in FH and normal subjects in two major respects. Normals secrete greater than 90% of apoB as VLDL, while one-third of apoB is secreted as intermediate density lipoprotein IDL/LDL in FH. Normals lose 40-50% of apoB from plasma as VLDL/IDL, while FH subjects lose none, metabolizing all of apoB to LDL. In FH, there is also the known prolongation of LDL residence time. The leucine tracer, biosynthetically incorporated into plasma apoB, permits distinguishing the separate pathways by which the metabolism of apoB is channeled. ApoB synthesis and secretion require 1.3 h. ApoB is secreted by three routes: 1) as large VLDL where it is metabolized by a delipidation chain; 2) as a rapidly metabolized VLDL fraction converted to LDL; and 3) as IDL or LDL. ApoB is metabolized along two pathways. The delipidation chain processes large VLDL to small VLDL, IDL, and LDL. The IDL pathway channels nascent, rapidly metabolized VLDL and IDL particles into LDL. It thus provides a fast pathway for the entrance of apoB tracer into LDL, while the delipidation pathway is a slower route for channeling apoB through VLDL into LDL. LDL apoB is derived in almost equal amounts from both pathways, which feed predominantly into large LDL. Small LDL is a product of large LDL, and the major loss of LDL-apoB is from small LDL. Two features of apoB metabolism in FH, the major secretory pathway through IDL and the absence of a catabolic loss of apoB from VLDL/IDL, greatly facilitate measuring the metabolic channeling of apoB into LDL.     

Rapid turnover of apolipoprotein C-III-containing triglyceride-rich lipoproteins contributing to the formation of LDL subfractions

The atherogenicity theory for triglyceride-rich lipoproteins (TRLs; VLDL + intermediate density lipoprotein) generally cites the action of apolipoprotein C-III (apoC-III), a component of some TRLs, to retard their metabolism in plasma. We studied the kinetics of multiple TRL and LDL subfractions according to the content of apoC-III and apoE in 11 hypertriglyceridemic and normolipidemic persons. The liver secretes mainly two types of apoB lipoproteins: TRL with apoC-III and LDL without apoC-III. Approximately 45% of TRLs with apoC-III are secreted together with apoE. Contrary to expectation, TRLs with apoC-III but not apoE have fast catabolism, losing some or all of their apoC-III and becoming LDL. In contrast, apoE directs TRL flux toward rapid clearance, limiting LDL formation. Direct clearance of TRL with apoC-III is suppressed among particles also containing apoE. TRLs without apoC-III or apoE are a minor, slow-metabolizing precursor of LDL with little direct removal. Increased VLDL apoC-III levels are correlated with increased VLDL production rather than with slow particle turnover. Finally, hypertriglyceridemic subjects have significantly greater production of apoC-III-containing VLDL and global prolongation in residence time of all particle types. ApoE may be the key determinant of the metabolic fate of atherogenic apoC-III-containing TRLs in plasma, channeling them toward removal from the circulation and reducing the formation of LDLs, both those with apoC-III and the main type without apoC-III.     

ApoB-75, a truncation of apolipoprotein B associated with familial hypobetalipoproteinemia: genetic and kinetic studies.

We have identified a mutation of apolipoprotein B (apoB) in a kindred with hypobetalipoproteinemia. Four affected members had plasma concentrations of total cholesterol of 115 +/- 14, low density lipoprotein (LDL)-C of 48 +/- 11, and apoB of 28 +/- 9 (mg/dl mean +/- SD). The values correspond to approximately 30% the values for unaffected relatives. Triglyceride and high density lipoprotein (HDL)-C concentrations were 92 +/- 50 and 49 +/- 4, respectively, neither significantly different from unaffected relatives. Western blots of plasma apoB of affected subjects showed two major bands: apoB-100 and an apoB-75 (mol wt of approximately 418,000). DNA sequencing of the appropriate polymerase chain reaction (PCR)-amplified genomic DNA segment revealed a deletion of the cytidine at nucleotide position 10366, resulting in a premature stop codon at amino acid residue 3387. In apoB-75/apoB-100 heterozygotes, two LDL populations containing either apoB-75 or apoB-100 could be distinguished from each other by gel permeation chromatography and by immunoblotting of nondenaturing gels using monoclonal antibodies B1B3 (epitope between apoB amino acid residues 3506-3635) and C1.4 (epitope between residues 97-526). ApoB-75 LDL were smaller and more dense than apoB-100 LDL. To determine whether the low concentration of apoB-75 was due to its enhanced LDL-receptor-mediated removal, apoB-75 LDL were isolated from the proband's d 1.063-1.090 g/ml fraction (which contained most of the apoB-75 in his plasma) by chromatography on anti-apoB and anti-apoA-I immunoaffinity columns. The resulting pure apoB-75 LDL fraction interacted with the cells 1.5-fold more effectively than apoB-100 LDL (d 1.019-1.063 g/ml). To determine the physiologic mechanism responsible for the hypobetalipoproteinemia, in vivo kinetic studies were performed in two affected subjects, using endogenous labeling of apoB-75 and apoB-100 with [13C]leucine followed by multicompartmental kinetic analyses. Fractional catabolic rates of apoB-75 VLDL and LDL were 2- and 1.3-fold those of apoB-100 very low density lipoprotein (VLDL) and LDL, respectively. Production rates of apoB-75 were approximately 30% of those for apoB-100. This differs from the behavior of apoB-89, a previously described variant, whose FCRs were also increased approximately 1.5-fold relative to apoB-100, but whose production rates were nearly identical to those of apoB-100. Thus, in contrast to the apoB-89 mutation, the apoB-75 mutation imparts two physiologic defects to apoB-75 lipoproteins that account for the hypobetalipoproteinemia, diminished production and increased catabolism.     

Relationship between cholesteryl ester transfer protein and LDL heterogeneity in familial hypercholesterolemia

Small, dense LDL particles have been associated with an increased risk of coronary artery disease, and cholesteryl ester transfer protein (CETP) has been suggested to play a role in LDL particle remodeling. We examined the relationship between LDL heterogeneity and plasma CETP mass in familial hypercholesterolemia (FH). LDL particles were characterized by polyacrylamide gradient gel electrophoresis in a total of 259 FH heterozygotes and 208 nonFH controls. CETP mass was measured by enzyme-linked immunosorbent assay in a subgroup of 240 participants, which included 120 FH patients matched with 120 controls. As compared with controls, FH subjects had an 11% higher CETP mass. Moreover, LDL-peak particle diameter (LDL-PPD) was significantly smaller in FH heterozygotes than in controls (258.1 +/- 4.8 vs. 259.2 +/- 4.1 A; P = 0.01) after adjustment for covariates. There was also an inverse relationship between LDL-PPD and CETP mass (R = -0.15; P = 0.02), and this relationship was abolished by adjustment for the FH/control status, indicating that LDL-PPD changes in FH are mediated, at least in part, by an increase in plasma CETP mass concentrations. These results suggest that increased plasma CETP mass concentrations could lead to significant LDL particle remodeling in FH heterozygotes and could contribute to the pathogenesis of atherosclerosis.     

Defective catabolism and abnormal composition of low-density lipoproteins from mutant pigs with hypercholesterolemia

Metabolic and chemical properties of low-density lipoproteins (LDLs) were studied in a strain of pigs carrying a specific apo-B allele associated with hypercholesterolemia and premature atherosclerosis. LDL mass was significantly greater in mutant than in control pigs (400 +/- 55 mg/dL vs 103 +/- 26 mg/dL), as was LDL cholesterol. When normal and mutant LDLs were injected into the bloodstream of normal pigs, the fractional catabolic rate (FCR) of mutant LDL was about 30% lower than that of control LDL. In mutant pigs, the mean FCRs of mutant and control LDL were similar, although they were much lower than the corresponding FCRs observed in normal pigs. The density profile of LDL particles differed in control and mutant pigs; the peak LDL flotation rate was shifted from S0f = 5.3 +/- 1.9 in controls to a more buoyant 7.4 +/- 0.5 in mutants. The elevation of LDL in the mutants was restricted to the most buoyant LDL subspecies. This subpopulation of mutant LDL was enriched with cholesteryl ester (47% vs 37%) and depleted of triglyceride, relative to LDL of similar density and size in controls. The lipid compositions of the denser LDL subpopulations (rho greater than 1.043 g/mL) were similar in mutants and controls. We conclude that the hypercholesterolemia of these mutant pigs is accounted for by defective catabolism of LDL. The buoyant cholesterol ester enriched LDL subspecies that accumulate in plasma may contribute to the accelerated atherogenesis that occurs in these animals.     

Role of LDL subfraction heterogeneity in the reduced binding of low density lipoproteins to arterial proteoglycans in cynomolgus monkeys fed a fish oil diet.

Previous studies using cynomolgus monkeys have shown that isocaloric substitution of dietary fish oil for lard reduced the in vitro binding of plasma low density lipoproteins (LDL) to arterial proteoglycans (PG) (Edwards, I.J., A.K. Gebre, W. D. Wagner, and J. S. Parks. 1991. Arterioscler. Thromb., 11: 1778-1785). The purpose of the present study was to determine whether all LDL subfractions were equally affected by the type of dietary fat with regard to PG binding and to identify compositional changes in LDL subfractions that might relate to the differential in PG binding. Two groups of cynomolgus monkeys (n = 5 each) were fed atherogenic diets (40% calories as fat; 0.26 mg cholesterol/kcal) containing 20% of calories as egg yolk and 20% as either lard or menhaden fish oil. LDL were isolated from plasma by ultracentrifugation and size exclusion chromatography and subfractionated by density gradient centrifugation. Three density ranges of LDL subfractions were collected from the gradients for determination of chemical composition, apoE and apoB content by ELISA, and binding to arterial PG in vitro. The d 1.015-1.025 g/ml subfraction contained 39 +/- 8% of the LDL cholesterol in the lard group but only 7 +/- 3% for the fish oil group. Values for cholesterol distribution were opposite for the d 1.035-1.045 g/ml subfraction, 8 +/- 1% versus 41 +/- 8%, respectively. Similar trends were noted for the distribution of apoB. For the lard group, LDL binding to arterial PG increased with decreasing density (i.e., increasing size) of the subfractions.(ABSTRACT TRUNCATED AT 250 WORDS)