P. Alaupovic

Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase

Abstract A hyperlipoproteinemic patient was investigated for lipoprotein abnormalities as a result of an abnormally high proportion (30%) of triglyceride in low density lipoprotein (LDL), the presence of β-VLDL as well as a reduced post-heparin lipolytic activity. Analysis of the component lipase activities in the proband and his brother by selective inactivation with protamine or by affinity chromatography revealed that lipoprotein lipase was present in normal concentrations while the triglyceride lipase characteristic of that released from liver was reduced to 5% of normal levels. Although evidence was obtained to indicate inhibition, in vitro, of post-heparin lipolytic activity of a normal subject, analysis of the patients post-heparin plasma revealed that pre-β-VLDL was catabolized while β-VLDL- and TG-enriched LDL were poor substrates. Very low density lipoprotein (VLDL) was markedly elevated in the proband and his brother and possessed pre-β- and β-mobility on agarose gel electrophoresis. Apolipoproteins A-I, A-II and D were present in normal concentrations while apolipoproteins B, C-II, C-III and E were elevated 2–3-fold over normal concentrations. There was no deficiency of apolipoprotein E-III in VLDL which is usually characteristic of the accumulation of β-migrating VLDL in primary Type III hyperlipoproteinemia. Both LDL and high density lipoprotein (HDL) were enriched in triglyceride and phosphatidyl choline at the expense of cholesterol ester and sphingomyelin. Mass analysis and analytic ultracentrifugation indicated that HDL was elevated in these subjects in contrast to a tendency for decreased HDL in most hypertriglyceridemic subjects. It is proposed that the presence of LDL and HDL 2 , which are relatively enriched in triglyceride and phosphatidyl choline, are a consequence of a deficiency of hepatic lipase. The accumulation of β-VLDL may also be associated with this enzyme deficiency or possibly result from a lipoprotein lipase which may be less active in the presence of lipoprotein components accumulating as a result of the hepatic lipase deficiency.

Lack of effect of probucol on atheroma formation in cholesterol-fed rabbits kept at comparable plasma cholesterol levels

Rabbits were fed cholesterol for 14 weeks to study the effect of probucol on atheroma formation. Three groups of animals were investigated: group CHOL was fed 1% cholesterol and served as control for group P + CHOL. fed 1% cholesterol and 1% probucol from the onset till the end of the experiment: group CHOL + P received 1% cholesterol throughout the experiment and 1% probucol during the last 4 weeks only. Plasma cholesterol concentrations were monitored at frequent intervals and were modulated by dietary perturbations so that the areas under the curve expressing plasma cholesterol changes with time, were similar in probucol and non-treated rabbits. The efficacy of long-term probucol treatment was evidenced by a significant reduction in plasma apolipoprotein A-I throughout the experiment and lower plasma TBARs during the first 6 weeks, when the hypocholesterolemic effect of probucol was also seen. Two weeks prior to the termination of the experiment, the rabbits were injected with rabbit plasma labeled with [3H]cholesteryl linoleyl ether [( 3H]CLE). Aortic atheromatosis was quantified by determination of total and cholesteryl ester (CE). The aortic cholesterol content was related to the arch, thoracic and abdominal segments, to the surface area of each segment or its dry defatted weight. Total and esterified cholesterol were highest in the aortic arch in all 3 groups when related to any of the above mentioned parameters. No statistically significant difference in aortic total cholesterol and CE content was seen among the three groups studied. The [3H]CLE recovered in the aortic segment correlated with the CE content and the [3H]CLE (dpm)/mg CE in all segments was similar. No statistically significant difference in the [3H]CLE recovered in the aortic segments among the 3 groups was seen. We conclude that in cholesterol-fed rabbits, in which the plasma cholesterol levels were maintained at comparable levels, probucol treatment did not affect plasma CE influx into the aorta and did not attenuate development of aortic atherosclerosis.

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 microM) and colchicine (1 microM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.

Identification of the abnormal cholestatic lipoprotein (LP-X) in familial lecithin:Cholesterol acyltransferase deficiency.

Virtually all lipids of human plasma circulate in association with specific proteins to yield lipid-protein complexes or lipoproteins. The lipoproteins are conventionally classified into four main groups based on ultracentrifugal flotation or electrophoretic mobility: chylomicrons, very low density lipoprotein (VLDL) or pre+lipoprotein, low density lipoprotein (LDL) or @lipoprotein, and high density lipoprotein (HDL) or ol -lipoprotein. According to the chemical classification system [l] , plasma lipoproteins consist of a mixture of polydisperse lipoprotein families each of which is characterized by the presence of a single, distinct apolipoprotein or its constitutive poly peptides: lipoprotein family LP-A is characterized by apolipoprotein A, lipoprotein family LP-B by apelipoprotein B and lipoprotein family LP-C by ape lipoprotein C. In sera of normal subjects and most patients with hyperlipoproteinemia of different types, 60-80% of the cholesterol is esterified. However, the levels of esterified cholesterol have been reported to be very low in obstructive jaundice [2 ] and in familial lecithin:cholesterol acyltransferase (LCAT) deficiency [31-

Apolipoprotein and lipoprotein concentrations in familial apolipoprotein C-11 deficiency☆

Lipoprotein and apolipoprotein concentrations were determined in 11 homozygous and 9 heterozygous subjects for familial apolipoprotein C-II (Apo C-II) deficiency. Apo C-II was not detectable in the homozygotes, with the exception of 1 subject who possessed immunochemically detectable quantities in one of two samples. Apolipoproteins C-III (Apo C-III) and E (Apo E) were elevated 2-3-fold in 9 of 11 homozygotes. Apo C-III, but not Apo E, correlated with triglyceride levels (1500-4100 mg/dl). However, both Apo C-III and Apo E correlated with the cholesterol levels and one another. Apolipoproteins A-I (Apo A-I), A-II (Apo A-II) and B (Apo B) were reduced to approximately 50-60% of normal values in association with very low levels of cholesterol in high density (HDL; 11 +/- 2 mg/dl) and low density (LDL; 19 +/- 6 mg/dl) lipoproteins in the homozygous subjects. These alterations were associated with a marked decrease in the proportion of plasma Apo C-III associated with HDL. The levels of apolipoprotein D (Apo D) were within the normal range. Nine obligate heterozygotes had Apo C-II concentrations (mean 1.8 +/- 0.5 mg/dl; range 1.2-2.7 mg/dl) which were approximately 40-50% of normal values (mean 2.9 +/- 0.9 mg/dl; range 1.7-5.6 mg/dl). The reduction in absolute amounts of Apo C-II was also reflected in a reduction of the ratio Apo C-II/Apo C-III in very low density lipoproteins (VLDL) and in a reduction in the ability of the sera to activate skim milk lipoprotein lipase. The concentrations of Apo A-II, Apo B, Apo C-III and Apo E were normal. Apo A-I concentrations were normal or slightly low in association with slightly reduced concentrations of HDL cholesterol and a low proportion of plasma Apo C-III in HDL in relation to LDL and VLDL in some heterozygotes. It is concluded that the marked alterations in the apolipoprotein levels in homozygous subjects are primarily a reflection of the deficiency of Apo C-II which results in severe hypertriglyceridemia. In heterozygotes, the partial deficiency of Apo C-II appears to result in a minor disturbance of the clearance of the triglycerides and Apo C-III rich particles but no marked changes in the concentrations of total lipids, lipoproteins and apolipoproteins in fasting plasma.

Quantitative determination of human plasma apolipoprotein A-I by a noncompetitive enzyme-linked immunosorbent assay

A noncompetitive enzyme-linked immunosorbent assay (ELISA) for human plasma apolipoprotein A-I (ApoA-I) was developed. Microtiter plates were coated with purified antibodies to ApoA-I and blocked. Plasma samples from normolipidemic and hypertriglyceridemic subjects were added and ApoA-I was allowed to bind to coating antibodies. After washing, the amount of ApoA-I bound to microtiter plates was estimated with horseradish peroxidase-labeled antibodies to ApoA-I. A single step delipidization procedure was included to expose masked antigenic sites of ApoA-I in plasma. The average concentration of ApoA-I in plasma of normolipidemic subjects was 1.37 g/l. Recovery of ApoA-I added to plasma varied from 93-107%. Intra- and inter-assay coefficients of variations were 4 and 8%, respectively. The assay was also used for quantifying ApoA-I in lipoprotein density classes. There was a good correlation between this assay and electroimmunoassay (r = 0.84-0.92). The described sandwich ELISA is a specific, precise, sensitive and relatively simple method for measuring ApoA-I levels in human plasma.

Lipolytic degradation of human very low density lipoproteins by human milk lipoprotein lipase: The identification of lipoprotein B as the main lipoprotein degradation product☆

Although the direct conversion of very low density lipoproteins (VLDL) into low density (LDL) and high density (HDL) lipoproteins only requires lipoprotein lipase (LPL) as a catalyst and albumin as the fatty acid acceptor, the in vitro-formed LDL and HDL differ chemically from their native counterparts. To investigate the reason(s) for these differences, VLDL were treated with human milk LPL in the presence of albumin, and the LPL-generated LDL1-, LDL2-, and HDL-like particles were characterized by lipid and apolipoprotein composition. Results showed that the removal of apolipoproteins B, C, and E from VLDL was proportional to the degree of triglyceride hydrolysis with LDL2 particles as the major and LDL1 and HDL + VHDL particles as the minor products of a complete in vitro lipolysis of VLDL. In comparison with native counterparts, the in vitro-formed LDL2 and HDL + VHDL were characterized by lower levels of triglyceride and cholesterol ester and higher levels of free cholesterol and lipid phosphorus. The characterization of lipoprotein particles present in the in vitro-produced LDL2 showed that, as in plasma LDL2, lipoprotein B (LP-B) was the major apolipoprotein B-containing lipoprotein accounting for over 90% of the total apolipoprotein B. Other, minor species of apolipoprotein B-containing lipoproteins included LP-B:C-I:E and LP-B:C-I:C-II:C-III. The lipid composition of in vitro-formed LP-B closely resembled that of plasma LP-B. The major parts of apolipoproteins C and E present in VLDL were released to HDL + VHDL as simple, cholesterol/phospholipid-rich lipoproteins including LP-C-I, LP-C-II, LP-C-III, and LP-E. However, some of these same simple lipoprotein particles were present after ultracentrifugation in the LDL2 density segment because of their hydrated density and/or because they formed, in the absence of naturally occurring acceptors (LP-A-I:A-II), weak associations with LP-B. Thus, the presence of varying amounts of these cholesterol/phospholipid-rich lipoproteins in the in vitro-formed LDL2 appears to be the main reason for their compositional difference from native LDL2. These results demonstrate that the formation of LP-B as the major apolipoprotein B-containing product of VLDL lipolysis only requires LPL as a catalyst and albumin as the fatty acid acceptor. However, under physiological circumstances, other modulating agents are necessary to prevent the accumulation and interaction of phospholipid/cholesterol-rich apolipoprotein C- and E-containing particles.

Lipids and apolipoprotein profiles in men with aneurysmal and stenosing aorto-lliac atherosclerosis

The plasma lipids, lipoproteins and apolipoproteins have been compared in two groups of men with aorto-iliac atherosclerosis: Aneurysmal disease (n = 42) and stenosing disease (n = 86). The mean age of the men aneurysmal disease was 67.5 +/- 5.8 years and the mean age of the men with stenosing disease was 65.0 +/- 6.1 years: There was no significant different in body mass indices or smoking habits between the groups. The patients with aneurysmal disease had lower levels of plasma cholesterol than patients with stenosing disease (5.53 +/- 1.17 versus 6.11 +/- 1.20 mmol/L, P less than 0.05), but carried more cholesterol in VLDL compared to patients with stenosing disease (1.00 +/- 0.90 versus 0.60 +/- 65 mmol/L, P less than 0.05). Significantly lower concentration of apolipoprotein AI and HDL-cholesterol in patients with aneurysmal disease (ApoAI 1.01 +/- 0.31 versus 1.18 +/- 0.31 mmol/L, P less than 0.02, HDL 0.93 +/- 0.53 versus 1.13 +/- 0.34, P less than 0.05) was another characteristic difference between these two groups of patients with peripheral arterial disease. Otherwise, there were no obvious differences in the levels of plasma triglyceride, VLDL-triglyceride, LDL-cholesterol, and apolipoproteins B, C-III and E between the two groups. Although lipid and apolipoprotein profiles may not discriminate between aneurysmal and stenosing disease, different types of lipoprotein particles may contribute to the atherosclerotic process characterising both diseases.

Lipoprotein Particles in Hypertriglyceridemic States

The compositional and metabolic heterogeneity of operationally defined plasma lipoprotein classes (1-3) has necessitated the introduction of a classification system that utilizes apolipoproteins as specific markers for identifying and distinguishing discrete lipoprotein particles (1,4). In this system, lipoprotein particles are characterized and defined by their apolipoprotein composition (1,4). Studies on the quantification and distribution of apolipoproteins (4,5) have shown that apolipoprotein (Apo)B and ApoA (A-I 4- A-II) form two major groups of plasma lipoproteins. These two major lipoprotein groups may be separated (6) by immunoprecipitation or immunoaffinity chromatography of whole plasma (6). The use of these procedures results in the isolation of ApoA-containing lipoproteins free of ApoB. The fractionation of ApoA-containing lipoproteins into two major discrete lipoprotein particles LP-A-I and LP-A-I:A-II by immunoaffinity chromatography on an immunosorber with polyclonal antibodies to ApoA-II has already been described by Cheung and Albers (7). To identify discrete lipoprotein particles of the ApoB group of lipoproteins, we have developed a procedure based on sequential immunoprecipitation of ApoB-containing lipoproteins with polyclonal antisera to apolipoproteins B, E, C-III and, if necessary, C-II and C-I (6,8). The fractionation of very low density (VLDL, d < 1.006 g/ml) and two subtractions of low density (LDL.., d = 1.006-1.019 g/ml; LDL?, d = 1.019-1.063 g/ml lipoproteins from normolipidemic subjects by sequential immunoprecipitation showed that each of these density classes consists of a mixture of distinct lipoprotein particles including cholesterol ester-rich LP-B and triglyceride- rich LP-B:C-I:C-II:C-III:E (LP-B:C:E) and LP-B:C-I: C-II:C-III (LP-B:C) particles (8). The LP-B:C:E family of particles in some normolipidemic and hypercholesterolemic subjects also contained varying amounts of LP-B:E particles. In addition, small amounts of LP-B:C-I:E, LP-B:C-II, LP-C-III and LP-E particles were detected in some but not all-subjects or density classes. Each of the major ApoB-containing families of particles was shown to represent a polydisperse system of particles heterogeneous with respect to size, hydrated density, and lipid/protein ratio, but homogeneous with respect to the qualitative apolipoprotein composition.

Apolipoprotein-B subclasses as acceptors of cholesteryl esters transferred by CETP

Background  Five apolipoprotein (apo)‐defined apoB‐containing lipoprotein (Lp) subclasses designated LpB, LpB:C, LpB:E, LpB:C:E and LpA‐II:B:C:D:E are present in human plasma. This study was to determine whether these subclasses functioned equally as acceptors of cholesteryl esters (CE) transferred from high‐density lipoproteins (HDL) by CE transfer protein in healthy subjects with normal and mildly increased plasma triglyceride (TG) levels.