In vivo turnover study demonstrates diminished clearance of lipoprotein(a) in hemodialysis patients.

Lipoprotein(a) (Lp(a)) consists of a low-density lipoprotein-like particle and a covalently linked highly glycosylated protein, called apolipoprotein(a) (apo(a)). Lp(a) derives from the liver but its catabolism is still poorly understood. Plasma concentrations of this highly atherogenic lipoprotein are elevated in hemodialysis (HD) patients, suggesting the kidney to be involved in Lp(a) catabolism. We therefore compared the in vivo turnover rates of both protein components from Lp(a) (i.e. apo(a) and apoB) determined by stable-isotope technology in seven HD patients with those of nine healthy controls. The fractional catabolic rate (FCR) of Lp(a)-apo(a) was significantly lower in HD patients compared with controls (0.164+/-0.114 vs 0.246+/-0.067 days(-1), P=0.042). The same was true for the FCR of Lp(a)-apoB (0.129+/-0.097 vs 0.299+/-0.142 days(-1), P=0.005). This resulted in a much longer residence time of 8.9 days for Lp(a)-apo(a) and 12.9 days for Lp(a)-apoB in HD patients compared with controls (4.4 and 3.9 days, respectively). The production rates of apo(a) and apoB from Lp(a) did not differ significantly between patients and controls and were even lower for patients when compared with controls with similar Lp(a) plasma concentrations. This in vivo turnover study is a further crucial step in understanding the mechanism of Lp(a) catabolism: the loss of renal function in HD patients causes elevated Lp(a) plasma levels because of decreased clearance but not increased production of Lp(a). The prolonged retention time of Lp(a) in HD patients might importantly contribute to the high risk of atherosclerosis in these patients.

Differential metabolism of human VLDL according to content of ApoE and ApoC-III.

We studied the metabolism of very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) particles that did or did not have apolipoprotein E (apoE) in 12 normolipidemic women by endogenously labeling plasma apolipoprotein B. The plasma was separated into bound (E+) and unbound (E-) fractions by use of a monoclonal antibody (1D7), and the fractions were ultracentrifuged to yield E+ and E- subfractions of light and dense VLDL and IDL. VLDL E+ and IDL E+ were produced mainly by the liver. VLDL E+ and IDL E+ had lower fractional catabolic rates and much higher apolipoprotein C-III (apoC-III) content than did the corresponding E- particles. Most light VLDL apoE+ underwent lipolysis to dense VLDL E+ with reduced apoC-III content, which was removed from the circulation without conversion to IDL. In contrast, most light VLDL apoE-, poor in apoC-III, was removed from the circulation, and a smaller proportion underwent lipolysis to dense VLDL E-. Most dense VLDL E- underwent lipolysis to IDL E-. The rate constant for lipolysis of dense VLDL to IDL was greater for E- than for E+, and the rate constant for clearance from plasma was greater for dense VLDL E+ than for E-. In conclusion, metabolism of human VLDL particles is influenced by their content of apoE, further modulated by the coexistence of apoC-III.

Metabolic basis of high density lipoproteins and apolipoprotein A-I increase by HMG-CoA reductase inhibition in healthy subjects and a patient with coronary artery disease.

HMG-CoA reductase inhibitors, such as pravastatin, are widely used as lipid lowering drugs in hypercholesterolemia. Pravastatin does not only reduce the atherogenic low density lipoprotein (LDL)-cholesterol, but is also increasing high density lipoprotein (HDL)-cholesterol. However, the mechanism leading to an increase of HDL are unclear. Therefore, the effects of pravastatin on the in vivo kinetics of apolipoprotein (apo) A-I were studied in six normolipidemic subjects and in a patient with coronary artery disease (CAD) utilizing stable isotope tracer techniques. Two turnover studies were performed. The first turnover study was carried out before any drug treatment, the second study after 6 weeks of 40 mg pravastatin/day. Three times deuterium labeled L-leucine (3D-leucine) was given as a primed bolus constant infusion (bolus: 1340 microg/kg; infusion: 22 microg/kg per h), and tracer uptake into HDL apoA-I was determined by gas chromatography (GC)-mass-spectrometry (MS). In the healthy subjects HDL-cholesterol increased by 13% and apoA-I increased by 12% under pravastatin treatment. The HDL in the CAD patient decreased by 3% and apoA-I increased by 2%. Prior to drug treatment the mean apoA-I fractional synthetic rate (FSR) was 0.194 per day (S.D. +/- 0.02) and apoA-I production rate (PR) was 10.8 mg/kg per day (S.D. +/- 2.1). The CAD patient had a FSR of 0.219 per day and a PR of 10.6 mg/kg per day. After treatment with pravastatin the mean apoA-I FSR was 0.204 per day (S.D. +/- 0.02) and apoA-I PR was 12.5 mg/kg per day (S.D. +/- 1.5) in the healthy subjects. Despite only minor changes of HDL and apoA-I in the CAD patient, there were significant changes of FSR (0.267 per day) and PR (13.1 mg/kg per day) with pravastatin treatment. The in vivo kinetic data demonstrate an increased FSR of apoA-I. The increase in apoA-I is due to an increased PR of apoA-I. This study demonstrates increased production of HDL apoA-I as the metabolic cause of the increase in HDL and apoA-I levels under inhibition of HMG-CoA reductase in man.

Remnant-like very-low-density lipoprotein isolated from hypertriglyceridemic patients by immunoaffinity chromatography suppressed 3-hydroxy-3-methylglutaryl coenzyme A activity of cultured human skin fibroblasts.

It has been previously reported that VLDL unbound to monoclonal antibody against apoB-100 was rich in apoE, thus resembling remnant particles (J Lipid Res, 1993:33;369-380). In the current study, we have further analyzed the unbound VLDL fraction in plasma from hypertriglyceridemic patients using a mixture of monoclonal antibodies against apoB-100 and apoA-1. The unbound VLDL isolated from the plasma of hypertriglyceridemic patients was found to be rich in apoE, apoB-48, and triglyceride compared with the bound VLDL. Furthermore, these unbound VLDL, but not bound VLDL, significantly suppressed HMG CoA reductase activity of cultured human skin fibroblasts (-20 to -25%, P = 0.0022). The degree of suppression is significantly correlated with the apoE content of unbound VLDL (r = -0.769, P < 0.05). Unbound VLDL failed to suppress the activity of HMG CoA reductase of LDL receptor negative fibroblasts. These observations indicate a potential atherogenicity of remnant-like unbound VLDL by delivering more cholesterol through the LDL receptor dependent pathway with apoE as a ligand. In conclusion, this new immunoaffinity chromatography system is a useful method for directly quantifying atherogenic remnants in plasma.

Unravelling high density lipoprotein-apolipoprotein metabolism in human mutants and animal models.

Apolipoprotein A-I plays an essential structural and functional role in HDL metabolism and apolipoprotein A-II has important effects on HDL metabolism and function. Kinetic studies in humans have established that variation in plasma HDL-cholesterol and apolipoprotein A-I concentrations is primarily determined by variation in the rate of apolipoprotein A-I catabolism. In contrast, plasma apolipoprotein A-II levels are primarily determined by the rate of apolipoprotein A-II production. Genetic factors play an important role in modulating the plasma levels of HDL-cholesterol and apolipoproteins A-I and A-II. Studies in humans have established that mutations in genes encoding enzymes that esterify cholesterol (lecithin : cholesterol acyltransferase), transfer cholesterol (cholesteryl ester transfer protein) and hydrolyze lipids (hepatic lipase, lipoprotein lipase) regulate HDL-cholesterol and apolipoprotein A-I levels by modifying the lipid content (and therefore the size) of HDL particles. Recent studies in transgenic and knockout animals have confirmed the key role of HDL lipid-modifying proteins in HDL, apolipoprotein A-I and apolipoprotein A-II metabolism and have expanded our understanding of the role of lipid modification in determining plasma concentrations of HDL-cholesterol and apolipoprotein A-I, as well as the potential functional roles of apolipoprotein A-II.

ApoA-II kinetics in humans using endogenous labeling with stable isotopes: slower turnover of apoA-II compared with the exogenous radiotracer method.

ApoA-II is a major apolipoprotein constituent of high density lipoproteins (HDL) and may play an important role in lipoprotein metabolism and predisposition to atherosclerosis. Previous radiotracer kinetic studies have suggested that the metabolism of apoA-II in humans may be different than the metabolism of apoA-I, the major HDL apolipoprotein. In the present study, we have used an endogenous labeling technique using stable isotopically labeled amino acids to study apoA-II metabolism and compared the results to those obtained by a simultaneous exogenous radiotracer labeling method. Seven subjects with HDL cholesterol levels ranging from 9 to 93 mg/dl and apoA-II levels from 13 to 60 mg/dl were investigated in this study. [13C6]phenylalanine and 131I-labeled apoA-II were simultaneously administered as a primed-constant infusion and a bolus injection, respectively. In the endogenous labeling study, plateau tracer/tracee ratios of VLDL apoB-100 were used as estimates for the precursor pool tracer/tracee ratios for apoA-II synthesis. Residence times of apoA-II using these two independent methods were found to be highly correlated (r = 0.973, P < 0.0002). These results indicate that the endogenous labeling of apoA-II using stable isotopically labeled amino acids is a reasonable alternative to the conventional exogenous radiotracer labeling method for the investigation of apoA-II turnover. However, under the conditions of our experimental design and modeling strategy, the apoA-II residence times as determined by endogenous labeling were significantly longer (mean 5.33 days) than by exogenous radiotracer (mean 4.65 days). This suggests that apoA-II turnover may be even slower than believed based on radiotracer studies, and further supports the concept that HDL containing apoA-II are metabolized differently than HDL without apoA-II.

Increased catabolic rate of low density lipoproteins in humans with cholesteryl ester transfer protein deficiency.

The cholesteryl ester transfer protein (CETP) transfers lipids among lipoprotein particles and plays a central role in lipoprotein metabolism. Humans with genetic deficiency of CETP have both elevated HDL cholesterol and apolipoprotein A-I concentrations as well as decreased LDL cholesterol and apolipoprotein B levels. The present study was undertaken to elucidate the metabolic basis for the decreased LDL cholesterol and apo B levels in CETP deficiency. We conducted a series of in vivo apo B kinetic studies in tow unrelated homozygotes with CETP deficiency and in control subjects. A primed constant infusion of stable isotopically labeled phenylalanine was administered to the two CETP deficient subjects and control subjects and apo B kinetic parameters in VLDL, intermediate density lipoproteins, and LDL were obtained by using a multicompartmental model. The fractional catabolic rates (FCR) of LDL apo B were significantly increased in the CETP-deficient subjects (0.56 and 0.75/d) compared with the controls (mean FCR of 0.39/d). Furthermore, the production rates of apo B in VLDL and intermediate density lipoprotein were decreased by 55% and 81%, respectively, in CETP deficiency compared with the controls. In conclusion, CETP-deficient subjects were demonstrated to have substantially increased catabolic rates of LDL apo B as the primary metabolic basis for the low plasma levels of LDL apo B. This result indicates that the LDL receptor pathway may be up-regulated in CETP deficiency.

Apolipoprotein A-II production rate is a major factor regulating the distribution of apolipoprotein A-I among HDL subclasses LpA-I and LpA-I:A-II in normolipidemic humans.

HDLs are heterogeneous in their apolipoprotein composition. Apolipoprotein (apo) A-I and apoA-II are the major proteins found in HDL and form the two major HDL subclasses: those that contain only apoA-I (LpA-I) and those that contain both apoA-I and apoA-II (LpA-I:A-II). Substantial evidence indicates that these two subclasses differ in their in vivo metabolism and effect on atherosclerosis, with LpA-I the more specifically protective subfraction against atherosclerosis. The purpose of this study was to investigate the effect of apoA-I and apoA-II production and catabolism on plasma LpA-I and LpA-I:A-II levels. Fifty normolipidemic subjects (those with HDL cholesterol levels in the top and bottom tenth percentiles were excluded) underwent kinetic studies with radiolabeled apoA-I and apoA-II, and the kinetic parameters of apoA-I and apoA-II were correlated with LpA-I and LpA-I:A-II levels. ApoA-I levels were strongly correlated with apoA-I residence times and less strongly correlated with apoA-I production rates. In contrast, apoA-II levels were correlated only with apoA-II production rates and not with apoA-II residence times. Levels of apoA-I in LpA-I were correlated with apoA-I residence times, whereas levels of apoA-I in LpA-I:A-II were correlated primarily with apoA-II production rates. The fraction of apoA-I in LpA-I was highly inversely correlated with apoA-II production rate (r = -.67, P < .001). In multiple regression analysis, apoA-II production rate was the most significant independent variable determining percent apoA-I in LpA-I among all the kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of high-density apolipoprotein particles A-I and A-I:A-II isolated from humans with cholesteryl ester transfer protein deficiency.

The cholesteryl ester transfer protein (CETP) plays an important role in metabolism of high-density lipoprotein and reverse cholesterol transport in humans. The two major classes of high-density lipoprotein particles are those containing apolipoprotein A-I (LpA-I) and those containing both apoA-I and apoA-II (LpA-I:A-II). We isolated and characterized the apoA-I-containing lipoprotein particles from three subjects with homozygous CETP deficiency (CETP-D) and compared the results with those from normolipidemic control subjects. Plasma concentrations of apoA-I in both LpA-I and LpA-I:A-II were significantly elevated in CETP-D subjects. Both LpA-I and LpA-I:A-II from these subjects were larger and contained more cholesteryl ester per particle than control particles. In CETP-D, subpopulations of LpA-I and LpA-I:A-II with an unusually large size (Stokes diameters 13.8 nm and 12.6 nm, respectively) not detected in normal subjects were isolated. The molar ratio of apoA-I to apoA-II in LpA-I:A-II isolated from CETP-D subjects was higher (mean 2.4) than those of controls (mean 1.4). ApoE was primarily associated with LpA-I:A-II in CETP-D subjects. A subclass of LpA-I with pre-beta migration on agarose electrophoresis was increased in CETP-D subjects. Both LpA-I and LpA-I:A-II from CETP-D subjects bound with higher affinity but less capacity to HepG2 cells compared with control particles, and were internalized to a lesser extent than control particles. These data suggest that the absence of CETP in humans significantly affects the plasma concentration, size, composition, and cellular interaction of both major classes of apoA-I-containing lipoprotein particles.

In vivo metabolism of apolipoproteins A-I and E in patients with abetalipoproteinemia: implications for the roles of apolipoproteins B and E in HDL metabolism.

The metabolism of high density lipoproteins (HDL) is tightly linked to the metabolism of apoB-containing lipoproteins through the exchange and transfer of lipids and apolipoproteins within the plasma compartment. Abetalipoproteinemia (ABL), a genetic disease in which apoB is absent from the plasma and HDL are the sole plasma lipoproteins, is a model for the investigation of HDL metabolism without modification by apoB-containing lipoproteins. Apolipoproteins A-I and E are two of the major apolipoproteins in HDL. Plasma apoA-I levels, but not apoE levels, have been reported to be decreased in patients with ABL. Furthermore, HDL from ABL patients is enriched in apoE compared with normal subjects. The purpose of the present study was: 1) to elucidate the metabolic basis of the low apoA-I levels in ABL; 2) to determine whether in vivo apoE production rates are normal in the absence of apoB-lipoprotein secretion; and 3) to test the hypothesis that apoE influences apoA-I and HDL catabolism in ABL. 131I-labeled apoA-I and 125I-labeled apoE were reassociated with autologous lipoproteins and injected into two unrelated ABL patients and control subjects. The mean residence time of apoA-I in ABL (2.4 days) was significantly decreased by nearly 50% compared with control subjects (4.7 +/- 0.6 days). ApoA-I production rates were also significantly decreased by 40% in ABL (7.1 mg/kg-d) compared with control subjects (11.8 +/- 1.7 mg/kg-d). The mean residence time of apoE in ABL (0.50 days) was somewhat shorter than that of control subjects (0.66 +/- 0.15 days), whereas the mean apoE production rate in ABL (2.14 mg/kg-d) was not substantially different from that of control subjects (1.55 +/- 0.62 mg/kg-d). HDL subfractions LpA-I and LpA-I:A-II were isolated using immunoaffinity chromatography. In contrast to the normal metabolism, apoA-I in LpA-I:A-II particles was catabolized at a faster rate than apoA-I in LpA-I, accounting for the greater decrease of plasma LpA-I:A-II relative to LpA-I in the ABL patients. HDL subfractions with and without apoE were also isolated using anti-apoE immunoaffinity chromatography. Labeled apoA-I in apoE-containing HDL was catabolized faster than that in HDL without apoE. Among the three different forms of apoE, the apoE monomer was catabolized at the fastest rate, the apoE homodimer at an intermediate rate, and the apoE-A-II heterodimer had the slowest rate of catabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

The inverse association of plasma lipoprotein(a) concentrations with apolipoprotein(a) isoform size is not due to differences in Lp(a) catabolism but to differences in production rate.

Lipoprotein(a) (Lp[a]) is an atherogenic lipoprotein which is similar in structure to low density lipoproteins (LDL) but contains an additional protein called apolipoprotein(a) (apo[a]). Apo(a) is highly polymorphic in size, and there is a strong inverse association between the size of the apo(a) isoform and the plasma concentration of Lp(a). We directly compared the in vivo catabolism of Lp(a) particles containing different size apo(a) isoforms to establish whether there is an effect of apo(a) isoform size on the catabolic rate of Lp(a). In the first series of studies, four normal subjects were injected with radio-labeled S1-Lp(a) and S2-Lp(a) and another four subjects were injected with radiolabeled S2-Lp(a) and S4-Lp(a). No significant differences in fractional catabolic rate were found between Lp(a) particles containing different apo(a) isoforms. To confirm that apo(a) isoform size does not influence the rate of Lp(a) catabolism, three subjects heterozygous for apo(a) were selected for preparative isolation of both Lp(a) particles. The first was a B/S3-apo(a) subject, the second a S4/S6-apo(a) subject, and the third an F/S3-apo(a) subject. From each subject, both Lp(a) particles were preparatively isolated, radiolabeled, and injected into donor subjects and normal volunteers. In all cases, the catabolic rates of the two forms of Lp(a) were not significantly different. In contrast, the allele-specific apo(a) production rates were more than twice as great for the smaller apo(a) isoforms than for the larger apo(a) isoforms. In a total of 17 studies directly comparing Lp(a) particles of different apo(a) isoform size, the mean fractional catabolic rate of the Lp(a) with smaller size apo(a) was 0.329 +/- 0.090 day-1 and of the Lp(a) with the larger size apo(a) 0.306 +/- 0.079 day-1, not significantly different. In summary, the inverse association of plasma Lp(a) concentrations with apo(a) isoform size is not due to differences in the catabolic rates of Lp(a) but rather to differences in Lp(a) production rates.

Markedly accelerated catabolism of apolipoprotein A-II (ApoA-II) and high density lipoproteins containing ApoA-II in classic lecithin: cholesterol acyltransferase deficiency and fish-eye disease.

Classic (complete) lecithin:cholesterol acyltransferase (LCAT) deficiency and Fish-eye disease (partial LCAT deficiency) are genetic syndromes associated with markedly decreased plasma levels of high density lipoprotein (HDL) cholesterol but not with an increased risk of atherosclerotic cardiovascular disease. We investigated the metabolism of the HDL apolipoproteins (apo) apoA-I and apoA-II in a total of five patients with LCAT deficiency, one with classic LCAT deficiency and four with Fish-eye disease. Plasma levels of apoA-II were decreased to a proportionately greater extent (23% of normal) than apoA-I (30% of normal). In addition, plasma concentrations of HDL particles containing both apoA-I and apoA-II (LpA-I:A-II) were much lower (18% of normal) than those of particles containing only apoA-I (LpA-I) (51% of normal). The metabolic basis for the low levels of apoA-II and LpA-I:A-II was investigated in all five patients using both exogenous radiotracer and endogenous stable isotope labeling techniques. The mean plasma residence time of apoA-I was decreased at 2.08 +/- 0.27 d (controls 4.74 +/- 0.65 days); however, the residence time of apoA-II was even shorter at 1.66 +/- 0.24 d (controls 5.25 +/- 0.61 d). In addition, the catabolism of apoA-I in LpA-I:A-II was substantially faster than that of apoA-I in LpA-I. In summary, genetic syndromes of either complete or partial LCAT deficiency result in low levels of HDL through preferential hypercatabolism of apoA-II and HDL particles containing apoA-II. Because LpA-I has been proposed to be more protective than LpA-I:A-II against atherosclerosis, this selective effect on the metabolism of LpA-I:A-II may provide a potential explanation why patients with classic LCAT deficiency and Fish-eye disease are not at increased risk for premature atherosclerosis despite markedly decreased levels of HDL cholesterol and apoA-I.

Very low high-density lipoproteins without coronary atherosclerosis.

Epidemiological studies have established that concentrations of plasma high-density lipoproteins (HDL) are inversely associated with premature atherosclerosis, but the physiological basis of this relationship remains unknown. We investigated 5 probands with very low plasma HDL. None had clinical or biochemical findings typical of the known genetic disorders with low HDL nor had evidence of premature coronary atherosclerosis by sensitive diagnostic methods. All 5 probands and the son of 1 of them had rapid catabolism of the HDL apolipoproteins A-I and A-II. These results indicate that not all people with low HDL are necessarily at risk of premature coronary heart disease and that further investigation is required before decisions can be made about their management.

Evaluation of apoA-I kinetics in humans using simultaneous endogenous stable isotope and exogenous radiotracer methods.

Apolipoprotein A-I is the major apolipoprotein constituent of high density lipoproteins (HDL). Methods used to investigate in vivo kinetics of apoA-I include exogenous labeling with radioiodine and endogenous labeling with stable isotopically labeled amino acids. We report here a direct comparison of these methods to determine the in vivo kinetics of apoA-I in four normal subjects. Purified apoA-I was labeled with 125I, reassociated with autologous plasma, and injected into study subjects. At the same time, [13C6]phenylalanine was administered as a primed constant infusion for up to 14 hours. The kinetic parameters of apoA-I were determined from the 125I-labeled apoA-I plasma curves. For the analysis of data from stable isotope studies, very low density lipoprotein (VLDL) apoB-100, VLDL apoB-48, and total apoA-I were isolated by ultracentrifugation and subsequent preparative NaDodSO4-PAGE, hydrolyzed, and derivatized. The tracer/tracee ratio was determined by gas chromatography-mass spectrometry. Monoexponential function analysis was used to determine the tracer/tracee curves of VLDL apoB-100 and VLDL apoB-48, and total apoA-I. The mean plateau tracer/tracee ratio of VLDL apoB-100 (primarily liver-derived) was 5.19%, whereas that of VLDL apoB-48 (intestinally derived) was only 3.74%. Using the VLDL apoB-100 plateau tracer/tracee ratio as the estimate of the precursor pool enrichment for apoA-I, the mean apoA-I residence time (RT) was 5.14 +/- 0.41 days, compared with 4.80 +/- 0.30 days for the exogenous labeling method. The apoA-I RTs using these two methods were highly correlated (r = 0.874).(ABSTRACT TRUNCATED AT 250 WORDS)

Increased production of apolipoprotein A-I associated with elevated plasma levels of high-density lipoproteins, apolipoprotein A-I, and lipoprotein A-I in a patient with familial hyperalphalipoproteinemia.

Familial hyperalphalipoproteinemia (FHA) is a heritable trait associated with elevated plasma concentrations of high-density lipoprotein (HDL) cholesterol and possibly with longevity and protection against coronary heart disease (CHD). The metabolic basis and molecular etiology of FHA have not been established in most kindreds. The proband of a kindred with FHA and possible longevity was found to have elevated plasma levels of HDL cholesterol, apolipoprotein (apo) A-I, and lipoproteins containing apo A-I without apo A-II (Lp A-I), but normal levels of apo A-II and lipoproteins containing apo A-I with apo A-II (Lp A-I:A-II). The in vivo kinetics of apo A-I and apo A-II were studied in the FHA proband and in control subjects using both exogenous radiotracer (125I-apo A-I and 131I-apo A-II) and endogenous stable isotope (primed constant infusion of 13C6-phenylalanine) labeling techniques. The production rate (PR) of apo A-I was markedly increased in the FHA subject (28.9 mg/kg.d) compared with the control subjects (12.0 +/- 2.1 mg/kg.d), whereas the apo A-II PR was not substantially increased. The primary sequence of the proband's apo A-I gene, including 1.2 kb of the 5'-flanking sequence, was normal. We conclude that a selective upregulation of apo A-I production is one metabolic cause of FHA, and results in high plasma concentrations of HDL cholesterol, apo A-I, and Lp A-I and possibly in protection from atherosclerotic CHD.

Delayed catabolism of high density lipoprotein apolipoproteins A-I and A-II in human cholesteryl ester transfer protein deficiency.

Deficiency of the cholesteryl ester transfer protein (CETP) in humans is characterized by markedly elevated plasma concentrations of HDL cholesterol and apoA-I. To assess the metabolism of HDL apolipoproteins in CETP deficiency, in vivo apolipoprotein kinetic studies were performed using endogenous and exogenous labeling techniques in two unrelated homozygotes with CETP deficiency, one heterozygote, and four control subjects. All study subjects were administered 13C6-labeled phenylalanine by primed constant infusion for up to 16 h. The fractional synthetic rates (FSRs) of apoA-I in two homozygotes with CETP deficiency (0.135, 0.134/d) were found to be significantly lower than those in controls (0.196 +/- 0.041/d, P < 0.01). Delayed apoA-I catabolism was confirmed by an exogenous radiotracer study in one CETP-deficient homozygote, in whom the fractional catabolic rate of 125I-apoA-I was 0.139/d (normal 0.216 +/- 0.018/d). The FSRs of apoA-II were also significantly lower in the homozygous CETP-deficient subjects (0.104, 0.112/d) than in the controls (0.170 +/- 0.023/d, P < 0.01). The production rates of apoA-I and apoA-II were normal in both homozygous CETP-deficient subjects. The turnover of apoA-I and apoA-II was substantially slower in both HDL2 and HDL3 in the CETP-deficient homozygotes than in controls. The kinetics of apoA-I and apoA-II in the CETP-deficient heterozygote were not different from those in controls. These data establish that homozygous CETP deficiency causes markedly delayed catabolism of apoA-I and apoA-II without affecting the production rates of these apolipoproteins.

In vivo metabolism of apolipoprotein A-I in a patient with homozygous familial hypercholesterolemia.

Familial hypercholesterolemia (FH), caused by a defect in the low density lipoprotein (LDL) receptor, results in high plasma concentrations of LDL cholesterol due to both overproduction and delayed catabolism of LDL. FH is also associated with significantly lower levels of plasma high density lipoprotein cholesterol and apolipoprotein (apo) A-I in both heterozygous and homozygous patients. However, the metabolic basis of the hypoalphalipoproteinemia in FH has not been elucidated. We investigated the kinetics of apo A-I in a homozygous FH patient and two normal control subjects by using endogenous labeling with a stable isotopically labeled amino acid. Study subjects were administered a primed constant infusion of 13C6-phenylalanine for 12 hours. Apolipoproteins were isolated from plasma drawn at selected time points and analyzed for their isotopic enrichment by gas chromatography-mass spectrometry. The fractional catabolic rate of apo A-I in the FH subject was found to be substantially increased (0.38 day-1) compared with that of the normal subjects (mean, 0.26 day-1). In addition, the apo A-I production rate was decreased in the FH subject (6.5 mg/kg.day-1) compared with the normal subjects (mean, 11.1 mg/kg.day-1). In conclusion, the low levels of high density lipoprotein cholesterol and apo A-I in this homozygous FH patient are due to the combined metabolic defects of increased apo A-I catabolism and decreased apo A-I production.

Counter-current chromatography of lipoproteins with a polymer phase system using the cross-axis synchronous coil planet centrifuge.

Lipoproteins were separated by counter-current chromatography using the type-XLL coil planet centrifuge. The separation was performed with a polymer phase system composed of 16% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 0.5 ml/min. About 5 ml of the sample solution containing approximately 150 mg of a lipoprotein mixture were loaded. High- and low-density lipoproteins were resolved within 12 h. Each component was detected by gel electrophoresis with oil red staining.