Studies on the metabolism of apolipoprotein B in hypertriglyceridemic subjects using simultaneous administration of tritiated leucine and radioiodinated very low density lipoprotein

To study the metabolic pathways of apolipoprotein B (apoB), a series of studies were carried out in which both radioiodinated very low density lipoproteins (VLDL) and tritiated leucine were simultaneously injected into three hypertriglyceridemic subjects. The appearance and disappearance of tritium activity in VLDL apoB, intermediate density lipoprotein (IDL) apoB, and low density lipoprotein (LDL) apoB were followed as was the disappearance of iodine activity from VLDL and the appearance and disappearance of iodine activity in IDL apoB and LDL apoB. It was found that a delipidation chain could describe the kinetics of both endogenously and exogenously labeled VLDL. A slow component of VLDL was necessary to fit the VLDL 131I-labeled apoB data and was consistent with the observed VLDL [3H]apoB kinetics. In addition, the estimated rate of conversion of VLDL apoB to LDL exceeded that which appeared to pass through the measured IDL pools, suggesting that a fraction of the IDL was not directly observed. It was also found that a higher percentage of VLDL 131I-labeled apoB was converted to LDL apoB than was VLDL [3H]apoB. To evaluate possible causes of this apparent anomaly, simultaneous examination of all kinetic data was performed. This exercise resulted in the resolution of removal pathways from multiple compartments in the VLDL delipidation chain and the conversion of slowly metabolized VLDL to IDL and LDL. The wide spectrum of this loss pathway indicates that previous estimates of VLDL apoB production rate using the radioiodinated methodology probably represent lower bounds for the true physiologic variable. It is important to note that these direct losses were apparent only when the combination of endogenous and exogenous labeling was used.     

Nonuniform radiolabeling of VLDL apolipoprotein B: implications for the analysis of studies of the kinetics of the metabolism of lipoproteins containing apolipoprotein B

Radiolabeling of whole lipoproteins or individual apolipoproteins has been an essential tool for the determination of the kinetics of apolipoprotein metabolism in vivo. Mathematical analysis of specific radioactivity (SA) or total radioactivity data has demonstrated the existence of significant complexity in the plasma decay curves of several apolipoproteins. Results obtained during development of methods to study the metabolism of apolipoprotein B (apoB) in very low density lipoprotein (VLDL) subclasses isolated according to flotation (Sf) rates from whole radiolabeled (d less than 1.006 g/ml) VLDL suggested nonuniform radiolabeling of apoB in the three Sf subclasses being studied. We therefore determined apoB SA in VLDL Sf subclasses in ten hypertriglyceridemic and five normal subjects. After radioiodination of apoB in whole VLDL, different apoB SA were found in Sf 400-100, Sf 100-60, and Sf 60-20. The pattern of labeling was quite variable among subjects. On average, apoB SA in the VLDL tracer was greatest in Sf 400-100, and least in Sf 60-20. Nonuniform labeling could also be demonstrated in five studies in which samples were obtained 3 min after intravenous injection of the tracer into subjects with a wide range of plasma triglycerides. Nonuniform labeling of apoB in whole VLDL was also demonstrated in two of the subjects by isolating subclasses of their VLDL that did not bind to an anti-apolipoprotein E immunoaffinity column. These results indicate that the usual assumption of homogeneous labeling of apoB may be erroneous. We have derived a simple mathematical formula to study the consequences of this assumption in estimating kinetic parameters. It is shown that an erroneous assumption of homogeneous tracer labeling may significantly underestimate or overestimate the true production rate, even in a simple two-pool model. Identification of labeling characteristics and incorporation of this information into the mathematical analysis of the plasma radioactivity data can improve the accuracy of the analysis as well as the sensitivity of compartmental models generated by such data.     

Inhibition of ACAT by avasimibe decreases both VLDL and LDL apolipoprotein B production in miniature pigs.

An orally bioavailable acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, avasimibe (CI-1011), was used to test the hypothesis that inhibition of cholesterol esterification, in vivo, would reduce hepatic very low density (VLDL) apolipoprotein (apo) B secretion into plasma. ApoB kinetic studies were carried out in 10 control miniature pigs, and in 10 animals treated with avasimibe (10 mg/kg/d, n = 6; 25 mg/kg/d, n = 4). Pigs were fed a diet containing fat (34% of calories) and cholesterol (400 mg/d; 0.1%). Avasimibe decreased the plasma concentrations of total triglyceride, VLDL triglyceride, and VLDL cholesterol by 31;-40% 39-48%, and 31;-35%, respectively. Significant reductions in plasma total cholesterol (35%) and low density lipoprotein (LDL) cholesterol (51%) concentrations were observed only with high dose avasimibe. Autologous 131I-labeled VLDL, 125I-labeled LDL, and [3H]leucine were injected simultaneously into each pig and apoB kinetic data were analyzed using multicompartmental analysis (SAAM II). Avasimibe decreased the VLDL apoB pool size by 40;-43% and the hepatic secretion rate of VLDL apoB by 38;-41%, but did not alter its fractional catabolism. Avasimibe decreased the LDL apoB pool size by 13;-57%, largely due to a dose-dependent 25;-63% in the LDL apoB production rate. Hepatic LDL receptor mRNA abundances were unchanged, consistent with a marginal decrease in LDL apoB FCRs. Hepatic ACAT activity was decreased by 51% (P = 0.050) and 68% (P = 0.087) by low and high dose avasimibe, respectively. The decrease in total apoB secretion correlated with the decrease in hepatic ACAT activity (r = 0.495; P = 0.026).We conclude that inhibition of hepatic ACAT by avasimibe reduces both plasma VLDL and LDL apoB concentrations, primarily by decreasing apoB secretion.     

Lovastatin therapy reduces low density lipoprotein apoB levels in subjects with combined hyperlipidemia by reducing the production of apoB-containing lipoproteins: implications for the pathophysiology of apoB production

We investigated the metabolism of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) apolipoprotein B (apoB) in seven patients with combined hyperlipidemia (CHL), using 125I-labeled VLDL and 131I-labeled LDL and compartmental modeling, before and during lovastatin treatment. Lovastatin therapy significantly reduced plasma levels of LDL cholesterol (142 vs 93 mg/dl, P less than 0.0005) and apoB (1328 vs 797 micrograms/ml, P less than 0.001). Before treatment, CHL patients had high production rates (PR) of LDL apoB. Three-fourths of this LDL apoB flux was derived from sources other than circulating VLDL and was, therefore, defined as "cold" LDL apoB flux. Compared to baseline, treatment with lovastatin was associated with a significant reduction in the total rate of entry of apoB-containing lipoproteins into plasma in all seven CHL subjects (40.7 vs. 25.7 mg/kg.day, P less than 0.003). This reduction was associated with a fall in total LDL apoB PR and in "cold" LDL apoB PR in six out of seven CHL subjects. VLDL apoB PR fell in five out of seven CHL subjects. Treatment with lovastatin did not significantly alter VLDL apoB conversion to LDL apoB or LDL apoB fractional catabolic rate (FCR) in CHL patients. In three patients with familial hypercholesterolemia who were studied for comparison, lovastatin treatment increased LDL apoB FCR but did not consistently alter LDL apoB PR. We conclude that lovastatin lowers LDL cholesterol and apoB concentrations in CHL patients by reducing the rate of entry of apoB-containing lipoproteins into plasma, either as VLDL or as directly secreted LDL.     

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.     

Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells

Mutations in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) have been associated with autosomal dominant hypercholesterolemia. In vivo kinetic studies indicate that LDL catabolism was impaired and apolipoprotein B (apoB)-containing lipoprotein synthesis was enhanced in two patients presenting with the S127R mutation on PCSK9. To understand the physiological role of PCSK9, we overexpressed human PCSK9 in mouse and cellular models as well as attenuated the endogenous expression of PCSK9 in HuH7 hepatoma cells using RNA interference. Here, we show that PCSK9 dramatically impairs the expression of the low density lipoprotein receptor (LDLr) and, in turn, LDL cellular binding as well as LDL clearance from the plasma compartment in C57BL6/J mice but not in LDLr-deficient mice, establishing a definitive role for PCSK9 in the modulation of the LDLr metabolic pathway. In contrast to data obtained in S127R-PCSK9 patients presenting with increased apoB production, our study indicates that wild-type PCSK9 does not significantly alter the production and/or secretion of VLDL apoB in either cultured cells or mice. Finally, we show that unlike PCSK9 overexpression in mice, the S127R mutation in patients led to increased VLDL apoB levels, suggesting a potential gain of function for S127R-PCSK9 in humans.     

Metabolism of very low density lipoproteins in the pig. An in vivo study.

1. The metabolism of apolipoprotein B (apoB) was investigated in pigs injected with [125I]very low density lipoproteins (VLDL) to determine to which extent the two distinct low density lipoprotein subclasses (LDL1 and LDL2) derive from VLDL. 2. The lipoproteins were isolated by density gradient ultracentrifugation and the transfer of radioactivity from VLDL into LDL1 and LDL2 apoB was measured. 3. Only a minor portion of VLDL apoB was converted to LDL1 (7.7 +/- 3.2%) and LDL2 (3.6 +/- 1.5%), respectively. Thus, we conclude that the major portion of LDL, especially LDL2, is synthesized independently from VLDL catabolism.     

Abnormal in vivo metabolism of apoB-containing lipoproteins in human apoE deficiency

The present study was undertaken to elucidate the metabolic basis for the increased remnants and lipoprotein(a) [Lp(a)] and decreased LDL apolipoprotein B (apoB) levels in human apoE deficiency. A primed constant infusion of (13)C(6)-phenylalanine was administered to a homozygous apoE-deficient subject. apoB-100 and apoB-48 were isolated, and tracer enrichments were determined by gas chromatography-mass spectrometry, then kinetic parameters were calculated by multicompartmental modeling. In the apoE-deficient subject, fractional catabolic rates (FCRs) of apoB-100 in VLDL and intermediate density lipoprotein and apoB-48 in VLDL were 3x, 12x, and 12x slower than those of controls. On the other hand, the LDL apoB-100 FCR was increased by 2.6x. The production rate of VLDL apoB-100 was decreased by 45%. In the Lp(a) kinetic study, two types of Lp(a) were isolated from plasma with apoE deficiency: buoyant and normal Lp(a). (125)I-buoyant Lp(a) was catabolized at a slower rate in the patient. However, (125)I-buoyant Lp(a) was catabolized at twice as fast as (131)I-normal Lp(a) in the control subjects. In summary, apoE deficiency results in: 1) a markedly impaired catabolism of VLDL/chylomicron and their remnants due to lack of direct removal and impaired lipolysis; 2) an increased rate of catabolism of LDL apoB-100, likely due to upregulation of LDL receptor activity; 3) reduced VLDL apoB production; and 4) a delayed catabolism of a portion of Lp(a).     

Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry

The measurement of apolipoprotein B (apoB) in purified lipoproteins by immunological assays is subject to criticism because of denatured epitopes or immunoreactivity differences between purified lipoproteins and standard. Chemical methods have therefore been developed, such as the selective precipitation of apoB followed by quantification of the precipitate. In this study, we present the measurement of apoB concentration in lipoproteins purified by ultracentrifugation by combining isopropanol precipitation and gas chromatography/mass spectrometry. Very low density lipoprotein (VLDL; d < 1.006 g/mL); VLDL plus intermediate density lipoprotein (VLDL + IDL; d < 1.019 g/mL); and VLDL, IDL, and low density lipoprotein (VLDL + IDL + LDL; d < 1.063 g/mL) were purified by ultracentrifugation. Apolipoprotein B-100 was selectively precipitated by isopropanol. The leucine content of the pellet was then determined by gas chromatography/mass spectrometry, using norleucine as internal standard. Knowledge of the number of leucine molecules in one apoB-100 molecule makes it possible to calculate the plasma concentration of apoB in the various lipoprotein fractions. ApoB in IDL (d 1.006-1.019 g/mL) and LDL (d 1.019-1.063 g/mL) were then determined by subtracting VLDL-apoB from apoB in lipoproteins d < 1.019 and apoB in lipoproteins d < 1.019 g/mL from apoB in lipoproteins d < 1.063 g/mL, respectively. The isopropanol precipitate was verified as pure apoB (>97%) in lipoprotein fractions isolated from normo- and hyperlipidemic plasma and the method appeared reproducible.The combination of isopropanol precipitation and the GC/MS method appears therefore to be a precise and reliable method for kinetic and epidemiological studies.     

Metabolism of apoprotein B in selectively bred baboons with low and high levels of low density lipoproteins

Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) apoprotein (apo)-B turnover rates were measured simultaneously by injecting 131I-labeled VLDL and 125I-labeled LDL into fasting baboons (Papio sp.) selectively bred for high serum cholesterol levels and having either low or high LDL levels. The radioactivities in VLDL, intermediate density lipoprotein (IDL), LDL apoB, and urine were measured at intervals between 5 min and 6 days. Kinetic parameters for apoB were calculated in each baboon fed a chow diet or a high cholesterol, high fat diet (HCHF). VLDL apoB residence times were similar in the two groups of animals fed chow; they were increased by HCHF feeding in high LDL animals, but not in low LDL animals. Production rates of VLDL apoB were decreased by the HCHF diet in both high and low LDL animals. Most of the radioactivity from VLDL apoB was transferred to IDL. However, a greater proportion of radioactivity was removed directly from IDL apoB in low LDL animals than in high LDL animals, and only about one-third appeared in LDL. In high LDL animals, a greater proportion of this radioactivity was converted to LDL (61.4 +/- 7.2% in chow-fed animals and 49.2 +/- 10.9% in animals fed the HCHF diet; mean +/- SEM, n = 5). Production rates for LDL apoB were higher in high LDL animals than those in low LDL animals on both diets. The HCHF diet increased residence times of LDL apoB without changing production rates in both groups. VLDL apoB production was not sufficient to account for LDL apoB production in high LDL animals, a finding that suggested that a large amount of LDL apoB was derived from a source other than VLDL apoB in these animals.     

Microsomal triglyceride transfer protein -493T variant reduces IDL plus LDL apoB production and the plasma concentration of large LDL particles

The microsomal triglyceride transfer protein (MTP) is essential for the synthesis and secretion of apolipoprotein B (apoB)-containing lipoproteins. We investigated the role the MTP -493G/T gene polymorphism in determining the apoB-100 secretion pattern and LDL heterogeneity in healthy human subjects. Groups of carriers of the T and the G variants (n = 6 each) were recruited from a cohort of healthy 50-yr-old men. Kinetic studies were performed by endogenous [(2)H(3)]leucine labeling of apoB and subsequent quantification of the stable isotope incorporation. apoB production rates, metabolic conversions, and eliminations were calculated by multicompartmental modeling (SAAM-II). LDL subfraction distribution was analyzed in the entire cohort (n = 377). Carriers of the MTP -493T allele had lower plasma LDL apoB and lower concentration of large LDL particles [LDL-I: 136 +/- 57 (TT) vs. 175 +/- 55 (GG) mg/l, P < 0.01]. Kinetic modeling suggested that MTP -493T homozygotes had a 60% lower direct production rate of intermediate-density lipoprotein (IDL) plus LDL compared with homozygotes for the G allele (P < 0.05). No differences were seen in production rates of large and small VLDL, nor were there any differences in metabolic conversion or elimination rates of apoB between the genotype groups. This study shows that a polymorphism in the MTP gene affects the spectrum of endogenous apoB-containing lipoprotein particles produced in humans. Reduced direct production of LDL plus IDL appears to be related to lower plasma concentrations of large LDL particles.     

Synthesis and secretion of VLDL by rat hepatocytes--modulation by cholesterol and phospholipids.

The synthesis and secretion of apoB, the major protein component of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), were studied using rat hepatocytes maintained in primary culture. Supplementation of hepatocytes with rat serum VLDL and LDL increased the production of apoB while delipidated lipoproteins had no significant effect, suggesting a role for lipids in the production of apoB. Addition of cholesterol to the culture medium also increased the production of apoB in a concentration-dependent manner. Pulse labelling followed by chase in presence of cholesterol indicated enhancement in apoB secretion. Mevinolin which inhibits cholesterol synthesis significantly reduced the secretion of apoB. The presence of phosphatidylcholine and phosphatidylethanolamine in the culture medium also increased the secretion of apoB into the medium. These data suggest that availability of lipids, particularly cholesterol, is an important determinant of apoB synthesis and secretion as VLDL.     

Intrahepatic assembly of very low density lipoproteins. Rate of transport out of the endoplasmic reticulum determines rate of secretion

To identify the rate-limiting step(s) in the hepatic production of very low density lipoproteins (VLDL), we investigated the intracellular distribution and rate of intracellular transport of de novo synthesized apolipoprotein B (apoB). For all secretory proteins examined (i.e. albumin, large molecular weight apoB, and small molecular weight apoB) the rough and smooth microsomes contained the majority of intracellular de novo synthesized protein, while the Golgi subfraction contained 10% or less. Pulse-chase analysis of the intracellular movement of apoB and albumin showed that the first order rate constant (in terms of half-life) describing the rate of movement out of the smooth and rough microsomes determined the overall rate of movement out of the cell. These data suggest that movement out of the endoplasmic reticulum, the site where VLDL is assembled, determines the overall rate of secretion. Furthermore, compared to albumin, the rate of intracellular transport of apoB was approximately two times slower suggesting that processing unique to VLDL apoB occurring in the endoplasmic reticulum was responsible. Additional studies show that essentially all of the de novo synthesized 35S-labeled albumin (produced from a pulse of [35S]methionine) lost from the cell during the chase period could be recovered in the culture medium. In contrast, much less of large molecular weight apoB (36%) and small molecular weight apoB (60%) was recovered in the culture medium. Since these cultured rat hepatocytes do not take up or degrade newly secreted apoB, these data suggest that a significant amount of apoB is degraded intracellularly.     

Treatment with high-dose simvastatin reduces secretion of apolipoprotein B-lipoproteins in patients with diabetic dyslipidemia

HMG-CoA reductase inhibitors (statins) are effective lipid-altering drugs for the treatment of dyslipidemia in patients with type 2 diabetes mellitus. We conducted a randomized, double-blind, placebo-controlled, crossover design trial to determine the effects of simvastatin, 80 mg/day, on plasma lipid and lipoprotein levels and on the metabolism of apolipoprotein B (apoB) in VLDL, intermediate density lipoprotein (IDL), and LDL and of triglycerides (TGs) in VLDL. Simvastatin therapy decreased TG, cholesterol, and apoB significantly in VLDL, IDL, and LDL. These effects were associated with reduced production of LDL-apoB, mainly as a result of reduced secretion of apoB-lipoproteins directly into the LDL density range. Statin therapy also reduced hepatic production of VLDL-TG. There were no effects of simvastatin on the fractional catabolic rates of VLDL-apoB or -TG or LDL-apoB. The basis for decreased VLDL-TG secretion during simvastatin treatment is not clear, but recent studies suggest that statins may activate peroxisomal proliferator-activated receptor alpha (PPARalpha). Activation of PPARalpha could lead to increased hepatic oxidation of fatty acids and less synthesis of TG for VLDL assembly.     

Apolipoprotein E mediates binding of normal very low density lipoprotein to heparin but is not required for high affinity receptor binding

The relationship between the cholesteryl ester content of normal human very low density lipoprotein (VLDL) and its ability to bind to apolipoprotein E (apoE), heparin, and the low density lipoprotein (LDL) receptor have been compared. Plasma VLDL were separated by heparin affinity chromatography into two fractions: one with apoE and one without. Both fractions had the same cholesteryl ester content relative to apolipoprotein B (apoB). LDL, on the other hand, had a greater cholesteryl ester content. VLDL were modified by lipolysis to express the ability to bind apoE (Ishikawa, Y., Fielding, C. J., and Fielding, P. E. (1988) J. Biol. Chem. 263, 2744-2749). Lipolyzed VLDL with or without apoE were compared for their ability to bind to heparin or the up-regulated fibroblast LDL receptor. Lipolyzed VLDL bound with the same affinity to the receptor whether or not the particles contained apoE. ApoB, not apoE, appears then to be the important ligand for normal VLDL. On the other hand, modified VLDL without apoE, even though binding to the LDL receptor, did not bind to heparin. These data suggest that apoE mediates heparin binding in normal VLDL, that apoB mediates receptor binding, and that the cholesteryl ester content of VLDL is not a factor in the induction of the ability to bind apoE.     

Intracellular apoA-I and apoB distribution in rat intestine is altered by lipid feeding

Intracellular forms of chylomicrons, very low density lipoprotein (VLDL) and high density lipoprotein (HDL) have previously been isolated from the rat intestine. These intracellular particles are likely to be nascent precursors of secreted lipoproteins. To study the distribution of intracellular apolipoprotein among nascent lipoproteins, a method to isolate intracellular lipoproteins was developed and validated. The method consists of suspending isolated enterocytes in hypotonic buffer containing a lipase inhibitor, rupturing cell membranes by nitrogen cavitation, and isolating lipoproteins by sequential ultracentrifugation. ApoB and apoA-I mass are determined by radioimmunoassay and newly synthesized apolipoprotein characterized following [3H]leucine intraduodenal infusion. Intracellular chylomicron, VLDL, low density lipoprotein (LDL), and HDL fractions were isolated and found to contain apoB, and apoA-IV, and apoA-I. In the fasted animal, less than 10% of total intracellular apoB and apoA-I was bound to lipoproteins and 7% of apoB and 35% of apoA-I was contained in the d 1.21 g/ml infranatant. The remainder of intracellular apolipoprotein was in the pellets of centrifugation. Lipid feeding doubled the percentage of intracellular apoA-I bound to lipoproteins and increased the percentage of intracellular apoB bound to lipoproteins by 65%. Following lipid feeding, the most significant increase was in the chylomicron apoB and HDL apoA-I fractions. These data suggest that in the fasting state, 90% of intracellular apoB and apoA-I is not bound to lipoproteins. Lipid feeding shifts intracellular apolipoprotein onto lipoproteins, but most intracellular apolipoprotein remains non-lipoprotein bound. The constant presence of a large non-lipoprotein-bound pool suggests that apolipoprotein synthesis is not the rate limiting step in lipoprotein assembly or secretion.     

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.     

Mevinolin and cholestyramine inhibit the direct synthesis of low density lipoprotein apolipoprotein B in miniature pigs

Previous studies established that following simultaneous injection of 125I-labeled homologous very low density lipoproteins (VLDL) and 131I-labeled homologous low density lipoproteins (LDL) into miniature pigs, a large proportion of LDL apolipoprotein B (apoB) was synthesized directly, independent of VLDL or intermediate density lipoprotein (IDL) apoB catabolism. The possibility that cholestyramine alone (a bile acid sequestrant) or in combination with mevinolin (a cholesterol synthesis inhibitor) could regulate the direct LDL apoB synthetic pathway was investigated. 125I-labeled VLDL and 131I-labeled LDL were injected into miniature pigs (n = 8) during a control period and following 18 days of cholestyramine treatment (1.0 g kg-1d-1) or following 18 days of treatment with cholestyramine and mevinolin (1.2 mg kg-1d-1). ApoB in each lipoprotein fraction was selectively precipitated using isopropanol in order to calculate specific activity. In control experiments, LDL apoB specific activity curves reached their peak values well before crossing the VLDL or IDL apoB curves. However, cholestyramine treatment resulted in LDL apoB curves reaching maximal values much closer to the point of intersection with the VLDL or IDL curves. Kinetic analyses demonstrated that cholestyramine reduced total LDL apoB flux by 33%, which was due entirely to inhibition of the LDL apoB direct synthesis pathway since VLDL-derived apoB was unaffected. In addition, the LDL apoB pool size was reduced by 30% and the fractional catabolic rate of LDL apoB was increased by 16% with cholestyramine treatment. The combination of mevinolin and cholestyramine resulted in an even more marked inhibition of the direct LDL apoB synthesis pathway (by 90%), and in two animals this pathway was completely abolished. This inhibition was selective as VLDL-derived LDL apoB synthesis was not significantly different. LDL apoB pool size was reduced by 60% due primarily to the reduced synthesis as well as a 40% greater fractional removal rate. These results are consistent with the idea that cholestyramine and mevinolin increase LDL catabolism by inducing hepatic apoB, E receptors. We have now shown that the direct synthesis of LDL apoB is selectively inhibited by these two drugs.     

Effects of continuous conjugated estrogen and micronized progesterone therapy upon lipoprotein metabolism in postmenopausal women

The effects of continuously administering both conjugated equine estrogens (CEE) and micronized progesterone (MP) on the concentration, composition, production and catabolism of very low density (VLDL) and low density lipoproteins (LDL) have not previously been reported. The mechanism of the hormonally induced reductions of plasma LDL cholesterol of S(f) 0;-20 (mean 16%, P < 0.005) and LDL apoB (mean 6%, P < 0.025) were investigated by studying the kinetics of VLDL and LDL apolipoprotein (apo) B turnover after injecting autologous (131)I-labeled VLDL and (125)I-labeled LDL into each of the 6 moderately hypercholesterolemic postmenopausal subjects under control conditions and again in the fourth week of a 7-week course of therapy (0.625 mg/d of CEE + 200 mg/d of MP). The combined hormones significantly lowered plasma LDL apoB by increasing the mean fractional catabolic rate of LDL apoB by 20% (0. 32 vs. 0.27 pools/d, P < 0.03). Treatment also induced a significant increase in IDL production (6.3 vs. 3.7 mg/kg/d, P = 0.028). However, this did not result in an increase in LDL production because of an increase in IDL apoB direct catabolism (mean 102%, P = 0.033). VLDL kinetic parameters were unchanged and the concentrations of plasma total triglycerides (TG), VLDL-TG, VLDL-apoB did not rise as often seen with estrogen alone. Plasma HDL-cholesterol rose significantly (P < 0.02). Our major conclusion is that increased fractional catabolism of LDL underlies the LDL-lowering effect of the combined hormones.     

Effect of apolipoprotein E genotype on apolipoprotein B-100 metabolism in normolipidemic and hyperlipidemic subjects

The effect of apolipoprotein (apo) E genotype on apoB-100 metabolism was examined in three normolipidemic apoE2/E2, five type III hyperlipidemic apoE2/E2, and five hyperlipidemic apoE3/E2 subjects using simultaneous administration of ¹³¹I-VLDL and ¹²⁵I-LDL, and multi-compartmental modeling. Compared with normolipidemic apoE2/E2 subjects, type III hyperlipidemic E2/E2 subjects had increased plasma and VLDL cholesterol, plasma and VLDL triglycerides, and VLDL and intermediate density lipoprotein (IDL) apoB concentrations (P < 0.05). These abnormalities were chiefly a consequence of decreased VLDL and IDL apoB fractional catabolic rate (FCR). Compared with hyperlipidemic E3/E2 subjects, type III hyperlipidemic E2/E2 subjects had increased IDL apoB concentration and decreased conversion of IDL to LDL particles (P < 0.05). In a pooled analysis, VLDL cholesterol was positively associated with VLDL and IDL apoB concentrations and the proportion of VLDL apoB in the slowly turning over VLDL pool, and was negatively associated with VLDL apoB FCR after adjusting for subject group. VLDL triglyceride was positively associated with VLDL apoB concentration and VLDL and IDL apoB production rates after adjusting for subject group. A defective apoE contributes to altered lipoprotein metabolism but is not sufficient to cause overt hyperlipidemia. Additional genetic mutations and environmental factors, including insulin resistance and obesity, may contribute to the development of type III hyperlipidemia.