Apolipoprotein AIMilano. Partial lecithin:cholesterol acyltransferase deficiency due to low levels of a functional enzyme

The cholesterol esterification process was analyzed in 19 carriers of the apolipoprotein AIMilano (AIM) variant and in 19 age-sex matched controls by measuring lecithin:cholesterol acyltransferase (LCAT) mass, activity (i.e., cholesterol esterification with a standard proteoliposome substrate) and cholesterol esterification rate (i.e., cholesterol esterification in the presence of the endogenous substrate). The AIM subjects had lower LCAT mass (3.30 +/- 0.85 micrograms/ml), activity (71.1 +/- 36.4 nmol/ml per h) and cholesterol esterification rate (23.6 +/- 12.5 nmol/ml per h) compared to controls (5.22 +/- 0.74 micrograms/ml, 121.6 +/- 54.6 nmol/ml per h and 53.6 +/- 29.9 nmol/ml per h, respectively). The specific LCAT activity, i.e., LCAT activity per microgram of LCAT, was similar in the two groups, indicating that the LCAT protein in the AIM carriers is structurally and functionally normal. However, the specific cholesterol esterification rate was 23% lower in the AIM subjects (8.03 +/- 6.01 nmol/h per microgram) compared to controls (10.49 +/- 5.86 nmol/h per microgram; P less than 0.05). The capacity of HDL3, purified from both AIM and control plasma, to act as substrates for cholesterol esterification was similar, thus suggesting that other mechanism(s) may be in play. Carriers with a relative abundance of abnormal, small HDL3b particles had the most altered cholesterol esterification pattern. Upon evaluating all AIM subjects, a complex relationship between HDL structure, plasma lipid-lipoprotein levels and cholesterol esterification emerged, making the AIMilano condition a unique model for the study of the mechanisms regulating the cholesterol esterification-transfer process in man.     

Isolation and properties of porcine lecithin:cholesterol acyltransferase

Lecithin: cholesterol acyltransferase (LCAT, phosphatidylcholine: sterol O-acyltransferase, EC 2.3.1.43) was purified approximately 20 000-fold from pig plasma by ultracentrifugation, phenyl-Sepharose and hydroxyapatite chromatography. Purified LCAT had an apparent relative molecular mass of 69 000 +/- 2000. By isoelectrofocusing it separated into five or six bands with pI values ranging from pH 4.9 to 5.2. The amino acid composition was similar to that of the human enzyme. An antibody against pig LCAT was prepared in goat. The antibody reacted against pig LCAT and gave a reaction of partial identity with human LCAT. Incubation of pig plasma or purified enzyme with the antibody virtually inhibited LCAT activity. The same amount of antibody inactivated only 62% of the LCAT activity in human serum. Pig and human LCAT were activated to the same extent by either human or pig apolipoprotein A-I (apo-A-I) using small liposomes as substrate. Human apoA-I, however, caused a higher esterification rate for both enzymes. Using apoA-I and small liposomes as a substrate, the addition of apoC-II up to 4 micrograms/ml had no effect on the LCAT reaction, but above this concentration LCAT was inhibited. Small liposomes with phosphatidylcholine/cholesterol molar ratios of 3:1 up to 8.4:1 did not show any significant differences in the LCAT reaction, when used as substrates in the presence of various amounts of apoA-I and albumin. In contrast, the LCAT activity was significantly reduced by liposomes with phosphatidylcholine/cholesterol molar ratios below 3:1.     

Fractional cholesterol esterification rate and muscle cholesterol of healthy young men

A group of fourteen healthy young male volunteers was examined to define more exactly the relations between lecithin cholesterol acyltransferase activity (LCAT), fractional cholesterol esterification rate (FER), total cholesterol (TC) and its free and esterified fractions (FC, CE) in skeletal muscles under physiological conditions. The mean values (+/- S.D.) of LCAT activity (95.4 +/- 16.3 mumol .1(-1) per hour), and FER (7.45 +/- 1.54% per hour) corresponded to published data on normolipidaemic healthy men of normal body weight. The mean value of TC in muscles was 332 +/- 83 micrograms per 100 mg of non-collagen protein, of which 14 +/- 7.4 per cent was formed by cholesterol esters. There was positive correlation between TC in muscles and age. Significant positive correlations between FER and the content of esterified cholesterol in muscles, and between FER and the proportion of esterified to total muscle cholesterol were found. These results suggest a close interrelation of cholesterol ester metabolism in the plasma and in slow pool tissues.     

Determination of lecithin:cholesterol acyltransfer in mouse plasma and the influence of mercaptoethanol and sulphydryl blocking agents on its activity.

1. The cholesterol esterifying activity in mouse plasma has been identified as lecithin:cholesterol acyltransferase (LCAT) on the basis of stoichiometric data, predominant transfer of polyunsaturated fatty acids, wide pH optimum and inhibition of esterification by phospholipase A2 and sulphydryl blocking agents. The esterifying activity differed from that present in plasma of man, rat and other species since it was partially inhibited by mercaptoethanol and other thiols. 2. Stoichiometric correlations between unesterified cholesterol, lecithin and lysolecithin were not exact, suggesting possible involvement of other enzymes in the overall esterification process during in vitro incubation of mouse plasma. 3. The initial rate of cholesterol esterification was determined by in vitro incubation of mouse plasma, whose cholesterol had been labelled by prior in vivo injection of 3H-mevalonic acid. The mean rate was 281 +/- 74 nmol/ml/hr (mean +/- S.D., n = 12) and correlated with unesterified cholesterol concentration (r = 0.73, P less than 0.01).     

Reactivity of HDL subfractions towards lecithin-cholesterol acyltransferase. Modulation by their content in free cholesterol

(1) Human HDL2 (d 1.070-1.125) and HDL3 (d 1.125-1.21) labelled with unesterified [14C]cholesterol, were incubated with a source of lecithin-cholesterol acyltransferase. For optimal activity, the reaction required the addition of albumin in excess, at least 3-times greater than the concentration of HDL-free cholesterol. Under such conditions, the reaction appeared saturable. HDL3 was found the most efficient substrate and the Vmax values expressed for 1.5 IU LCAT/ml and with an albumin/free cholesterol ratio of 3, were 8.3 nmol free cholesterol esterified/ml per h and 4.1 nmol/ml per h for HDL3 and HDL2, respectively. (2) HDL3 were modified in the presence of VLDL by inducing triacylglycerol lipolysis with a semipurified lipoprotein lipase from bovine milk. The newly formed HDL had gained free cholesterol and phospholipids, so that about 50% of these modified HDL, referred to as light-LIP-HDL3, were reisolated in the HDL2 density range. Light-LIP-HDL3 were enriched mostly in free cholesterol (+ 160%) and in phospholipid (+ 40%). Their reactivity towards LCAT was half-reduced compared to parent HDL3, which correlated well with a decrease in their phospholipid/free cholesterol molar ratio. Moreover, HDL3 artificially enriched in free cholesterol and exhibiting a comparable PL/FC behaved like lipolysis-modified HDL in their reactivity towards LCAT. (3) HDL3 were also modified by co-incubation with VLDL (post-VLDL-HDL3), or with VLDL and a source of lipid transfer protein (CET-HDL3). The latter treatment greatly affected the lipid composition of the core particle (-25% esterified cholesterol, +190% TG). In both cases, the moderate decreasing LCAT reactivity observed could be related to the phospholipid/free cholesterol ratio. Thus, like in artificial substrates, the lipid composition of the HDL surface may control the rate of LCAT-mediated cholesterol esterification.     

Defective enzyme causes lecithin-cholesterol acyltransferase deficiency in a Japanese kindred

Lecithin-cholesterol acyltransferase mass levels and activity and apolipoproteins A-I, A-II, B and D were measured in a Japanese family who have a familial lecithin-cholesterol acyltransferase deficiency. This analysis was performed to gain insight into the molecular basis of the enzyme deficiency and to compare findings in this family with other families with familial lecithin-cholesterol acyltransferase deficiency. The mass of the enzyme in plasma was determined by a sensitive double antibody radioimmunoassay, and enzyme activity was measured by using a common synthetic substrate comprised of phosphatidylcholine, cholesterol and apolipoprotein A-I liposomes prepared by a cholate dialysis procedure. The lecithin-cholesterol acyltransferase-deficient subject had an enzyme mass level that was 35% of normal (2.04 micrograms/ml, as compared with an average normal level of 5.76 +/- 0.95 micrograms/ml in 19 Japanese subjects) and an enzyme activity of less than 0.1% of normal (0.07 nmol/h per ml, as compared with normal levels of 100 nmol/h per ml). This subject also had lower levels of apolipoproteins: apolipoprotein A-I was 53 mg/dl (42% of normal), apolipoprotein A-II was 10.6 mg/dl (31% of normal), apolipoprotein B was 68 mg/dl (68% of normal), and apolipoprotein D was 3.6 mg/dl (60% of normal). The three obligate heterozygotes had enzyme mass levels ranging from 65% to 100% of normal and enzyme activity levels ranging from 23% to 65% of normal (23.4, 56.8, and 64.7 nmol/h per ml, respectively). The proband's sister had an enzyme mass level of 6.55 micrograms/ml (114% of normal) and an enzyme activity of only 64.8 nmol/h per ml (65% of normal), suggesting that she was also a heterozygote for lecithin-cholesterol acyltransferase deficiency. The obligate heterozygotes and the sister had normal apolipoprotein levels. We conclude that the lecithin-cholesterol acyltransferase deficiency in this family is due to the production of a defective enzyme that is expressed in the homozygote as well as in the heterozygotes, and, further, that this family's mutation differs from that reported earlier for other Japanese lecithin-cholesterol acyltransferase-deficient families.     

Activation of lecithin-cholesterol acyltransferase by apolipoprotein D: comparison of proteoliposomes containing apolipoprotein D, A-I or C-I

To study the activation of lecithin-cholesterol acyl transferase (LCAT) (phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43) by apolipoprotein D in comparison to apolipoproteins A-I and C-I, proteoliposomes with a phosphatidylcholine/free cholesterol molar ratio of 24:1, containing 10-300 micrograms/ml of apolipoproteins were used. The proteoliposomes were prepared by the cholate dialysis technique. In all proteoliposome preparations we found rouleaux structures and stacked discs. The particles formed with apolipoprotein A-I were the most homogeneous, followed by apolipoprotein D- and apolipoprotein C-I-containing particles. Apolipoprotein A-I was the most potent LCAT activator in our system followed by apolipoproteins C-I and D. The fractional esterification rate observed with apolipoprotein D-containing substrates amounted to 15-48% that of apolipoprotein A-I-containing ones. Neither apolipoprotein A-I- nor C-I-containing proteoliposomes gave linear reaction kinetics with LCAT. Even during the first 15-30 min of incubation, the kinetics deviated strikingly from linearity at all apolipoprotein concentrations. In contrast, proteoliposomes containing apolipoprotein D exhibited linear reaction kinetics up to 60-90 min. At low apolipoprotein A-I concentrations (5 micrograms/ml), the addition of apolipoprotein D to the incubates resulted in significantly higher esterification rates as compared to substrates containing apolipoprotein A-I only. This was not the case using substrates with high apolipoprotein A-I concentrations (50 micrograms/ml). From our results we speculate that apolipoprotein D may have some stabilizing effect on the enzyme LCAT.     

New substrate for determination of serum lecithin:cholesterol acyltransferase

Serum lecithin:cholesterol acyltransferase (LCAT) was estimated by enzymatically measuring the decrease in unesterified cholesterol after incubation of serum with liposomes. A high-performance liquid chromatography (HPLC) study showed the uptake of the lipids of liposomes by serum high density lipoprotein. Of all the examined liposomes prepared from cholesterol and various synthetic phosphatidylcholines, liposomes with dimyristoylphosphatidylcholine (DMPC) were found to be the most reactive in the LCAT reaction. When serum was used as an enzyme source, addition of purified apolipoprotein A-I, which is known to be an endogenous activator of LCAT, to the assay mixture resulted in a slight decrease in enzyme activity. Using DMPC-cholesterol liposomes as the substrate, the LCAT activities in 120 human sera showed a mean value of 485.4 +/- 64.6 nmol/hr per ml (mean +/- SD), which is 4.4- to 5.4-fold higher than the values obtained by self-substrate methods. LCAT activity was a linear function of the serum sample volume up to 670 nmol/hr per ml and coefficients of variation (CV) less than 4% were obtained under the standardized conditions. Moreover, when partially purified LCAT was added to various heat-inactivated sera, the activity was efficiently recovered. These results suggest that this method is sensitive, reproducible, and not greatly influenced by serum components.     

The reactivity of plasma phospholipids with lecithin:cholesterol acyltransferase is decreased in fish oil-fed monkeys

The size of low density lipoproteins (LDL) is strongly correlated with LDL cholesteryl ester (CE) content and coronary artery atherosclerosis in monkeys fed cholesterol and saturated fat. African green monkeys fed 11% (weight) fish oil diets have smaller LDL and less CE per LDL particle than lard-fed animals. We hypothesized that this might be due to a lower plasma lecithin:cholesterol acyltransferase (LCAT) activity in fish oil-fed animals. Using recombinant particles made of egg yolk lecithin-[14C]cholesterol-apoA-I as exogenous substrate, we found no difference in plasma LCAT activity (27 versus 28 nmol CE formed per h/ml) of fish oil- versus lard-fed animals, respectively; furthermore, no diet-induced difference in immunodetectable LCAT was found. However, plasma phospholipids from fish oil-fed animals were over 4-fold enriched in n-3 fatty acids in the sn-2 position compared to those of lard-fed animals. Additionally, the proportion of n-3 fatty acid-containing CE products formed by LCAT, relative to the available n-3 fatty acid in the sn-2 position of phospholipids, was less than one-tenth of that for linoleic acid. The overall rate of LCAT-catalyzed CE formation with phospholipid substrates from fish oil-fed animals was lower (5-50%) than with phospholipid substrates from lard-fed animals. These data show that n-3 fatty acids in phospholipids are not readily utilized by LCAT for formation of CE; rather, LCAT preferentially utilizes linoleic acid for CE formation. The amount of linoleic acid in the sn-2 position of plasma phospholipids is reduced and replaced with n-3 fatty acids in fish oil-fed animals. As a result, LCAT-catalyzed plasma CE formation in vivo is likely reduced in fish oil-fed animals contributing to the decreased cholesteryl ester content and smaller size of LDL particles in the animals of this diet group.     

Evidence that lipid hydroperoxides inhibit plasma lecithin:cholesterol acyltransferase activity

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. Recently, we found that polar lipids isolated from minimally oxidized LDL produced a dramatic inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, suggesting that HDL-cholesterol transport may be impaired during early atherogenesis. In this study, we have identified molecular species of oxidized lipids that are potent inhibitors of LCAT activity. Treatment of LDL with soybean lipoxygenase generated small quantities of lipid hydroperoxides (20 +/- 4 nmol/mg LDL protein, n = 3); but when lipoxygenase-treated LDL (1 mg protein/ml) was recombined with the d > 1.063 g/ml fraction of human plasma, LCAT activity was rapidly inhibited (25 +/- 4 and 65 +/- 16% reductions by 1 and 3 h, respectively). As phospholipid hydroperoxides (PL-OOH) are the principal oxidation products associated with lipoxygenase-treated LDL, we directly tested whether PL-OOH inhibited plasma LCAT activity. Detailed dose-response curves revealed that as little as 0.2 and 1.0 mole % enrichment of plasma with PL-OOH produced 20 and 50% reductions in LCAT activity by 2 h, respectively. To gain insight into the mechanism of LCAT impairment, the enzyme's free cysteines (Cys31 and Cys184) and active site residues were "capped" with the reversible sulfhydryl compound, DTNB, during exposure to either minimally oxidized LDL or PL-OOH. Reversal of the DTNB "cap" after such exposures revealed that the enzyme was completely protected from both sources of peroxidized phospholipids. We, therefore, conclude that PL-OOH inhibited plasma LCAT activity by modifying the enzyme's free cysteine and/or catalytic residues. These studies are the first to suggest that PL-OOH may accelerate the atherogenic process by impairing LCAT activity.     

Serum activity and hepatic secretion of lecithin:cholesterol acyltransferase in experimental hypothyroidism and hypercholesterolemia

Lecithin:cholesterol acyltransferase (LCAT), the major cholesterol esterifying enzyme in plasma, plays an important role in the removal of cholesterol from peripheral tissues. This study in rat focuses upon the effects of hypothyroidism and cholesterol feeding on serum activity and hepatic LCAT secretion. To obviate the effect that inclusion of high concentrations of cholesterol in the rat serum may have on the proteoliposome used in the assay of LCAT, very low and low density lipoproteins (VLDL and LDL) were removed by ultracentrifugation at d 1.063 g/ml. The molar esterification rate in the euthyroid VLDL + LDL-free serum was found to be 0.94 +/- 0.06 compared to 0.67 +/- 0.05 in hypothyroid rats and 1.56 +/- 0.14 in hypercholesterolemic rats. LCAT secretion by suspension cultures of hepatocytes from hypercholesterolemic rats was found to be significantly depressed when compared to that for euthyroid and hypothyroid animals. Secretion by hepatocytes from hypothyroid rats was depressed for the first 0-4 hr, but rapidly recovered. The depressed secretion of LCAT by hepatocytes from hypercholesterolemic rats correlates with the appearance in the media of apoE-rich, discoidal HDL. Discoidal HDL was six times more effective as a substrate for purified human LCAT than HDL from hypercholesterolemic serum, and twice as effective as serum and nascent HDL from euthyroid animals. It is concluded that the depressed LCAT activity in serum from hypothyroid rats is due to a depressed hepatic secretion of the enzyme and that the elevated serum activity of hypercholesterolemic rats may be related to a defect in LCAT clearance. Finally, the appearance of discoidal HDL in the medium upon culture of hepatocytes from hypercholesterolemic rats appears to be due to an inhibition of LCAT secretion by these cells.     

Plasma lecithin-cholesterol acyltransferase activity and cholesterol and phospholipid levels in premature newborn infants

Lecithin-cholesterol acyltransferase (LCAT) activity has been suggested to play an important role in the regulation of lipid metabolism. The present study was undertaken to examine any relationship between LCAT activity and altered cholesterol levels in plasma of full-term and preterm newborn infants. Plasma total, free and esterified cholesterol, total phospholipid and LCAT activity (cholesterol esterified, nmol/ml per h) were determined in placental cord blood. There was a significant negative relationship between total cholesterol levels and gestational age. The increased cholesterol with prematurity was due to both free and esterified cholesterol. There was also a significant negative relationship between LCAT activity and free cholesterol levels but not between LCAT activity and total cholesterol and esterified cholesterol levels. There was no relationship between esterified-to-free cholesterol ratio and LCAT activity. Total phospholipid was not significantly related to either gestational age or LCAT activity. This study suggests that reduced LCAT activity may be one of the factors that result in the accumulation of cholesterol in premature infants.     

Familial lecithin-cholesterol acyltransferase deficiency in a Japanese family: Evidence for functionally defective enzyme in homozygotes and obligate heterozygotes

Summary Lecithin-cholesterol acyltransferase (LCAT) mass and activity were measured in a Japanese family with familial LCAT deficiency. The two LCAT-deficient subjects had LCAT mass approximately 40–46% of normal (2.65 and 2.31 g/ml respectively, as compared with normal levels of 5.76±0.95 g/ml in 19 Japanese subjects) and enzyme activity less than 10% of normal (9.1 and 8.3 nmol/h/ml respectively, as compared with normal levels of 100 nmol/h/ml). All obligate heterozygotes examined, including the father of the two LCAT-deficient subjects, and all five children of the deficient subjects had LCAT mass approximately 72–80% of the normal LCAT mass (4.12, 4.38, 4.45, 4.48, 4.49, 4.61 g/ml, respectively) and LCAT activity approximately half normal (51.9, 52.4, 54.2, 56.6, and 57.2 nmol/h/ml). We conclude that the two LCAT-deficient subjects of this family have functionally defective enzyme. Furthermore, the data suggest that the plasma of the obligate heterozygotes contain both normal and functionally defective enzymes.     

Factors influencing the cholesterol esterification rate in lecithin cholesterol acyltransferase radioassay

Factors affecting the esterification rate of cholesterol by lecithin cholesterol acyltransferase (LCAT E.C. 2.3.1.43) in native cold labelled substrates (human, rabbit, rat serum, plasma, VLDL, LDL depleted serum, rabbit intraocular fluids) repaired by use of ready-made 14C-cholesterol discs (Cholesterol kinetics LCAT-test, UVVVR, Czechoslovakia) were investigated. EDTA added to the serum during the cold incubation (18 h, 0 degrees C-4 degrees C) increased the rate of esterification due to elimination of Ca2+ ions. The similar stimulating effect was found in the presence of mercaptoethanol (ME) in the serum, while in the plasma already stimulated by EDTA no additional effect by ME could be noticed. Freezing and thawing did not affect the fractional esterification rate (FER-per cent of total serum unesterified cholesterol esterified per hour) in normolipidaemic sera, whereas in hyperlipidaemic sera, particularly those with high levels of VLDL, FER was stimulated. Esterification partially proceeded during the cold incubation of serum or plasma with 14C-cholesterol ready-to-use discs, attaining the values of about 0.3%/h and 2-6%/h, respectively, in human sera and in rabbit and rat sera. The starting level of esterification did not affect the linearity of LCAT reaction during warm incubation (30 min at 37 degrees C), neither was the absolute value of FER changed as compared with cold labelled sera with those inhibited by DTNB and reactivated by ME. Substantial LCAT activity was also detected in extremely diluted substrates--such as intraocular fluid collected from rabbits with induced uveitis or after preceding paracentesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Familial lecithin-cholesterol acyltransferase: Identification of heterozygotes with half-normal enzyme activity and mass

Summary Lecithin-cholesterol acyltransferase (LCAT) mass and activity were measured in a Canadian kindred of Italian and Swedish descent with familial LCAT deficiency. Four subjects had LCAT mass of 5.21±0.87 g/ml (mean±SD) and LCAT activity of 98.8±12.0 nmol/h/ml, well within their respective normal ranges. Five family members, including the parents, the maternal grandmother, and two of four siblings of the LCAT deficient subjects, had enzyme mass (2.85±0.32 g/ml) and activity (50.8±6.3 nmol/h/ml) approximately one-half that of normal levels. These presumed heterozygotes had normal levels of apolipoproteins A-I, A-II, B and D. The two subjects with LCAT deficiency had no detectable LCAT mass (below 0.1 g/ml) or LCAT activity (below 0.76 nmol/h/ml), apolipoprotein A-I and D levels approximately 50% of normal, and apolipoproteins B and A-II levels only 30–35% of normal. LCAT deficiency in this family is determined by an autosomal recessive mode. Furthermore, LCAT levels and activity are determined by two autosomal codominant alleles, LCATⁿ, the normal LCAT gene, and LCAT^d, the LCAT deficiency gene.     

Impaired cholesterol esterification in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy

Cholesterol esterification was examined in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy by incubating cells pretreated without fetal calf serum for 48h, with (14C) cholesterol for 24h. Impaired cholesterol esterification was found in these cells and free cholesterol was accumulated in plasma membrane and Golgi fractions. This impairment was also induced in control cells by adding leupeptin (20 micrograms/ml) or monensin (2 micrograms/ml). These findings suggest the importance of the role of lysosomes for esterification of cholesterol and give a hint as to the basic defect in type C Niemann-Pick disease.     

Lecithin:cholesterol acyltransferase-induced transformation of HepG2 lipoproteins

Previous studies with the human hepatoblastoma-derived HepG2 cell line in this laboratory have shown that these cells produce high density lipoproteins (HDL) that are similar to HDL isolated from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Experiments were, therefore, performed to determine whether HepG2 HDL could be transformed into plasma-like particles by incubation with LCAT. Concentrated HepG2 lipoproteins (d less than 1.235 g/ml) were incubated with purified LCAT or lipoprotein-deficient plasma (LPDP) for 4, 12, or 24 h at 37 degrees C. HDL isolated from control samples possessed excess phospholipid and unesterified cholesterol relative to plasma HDL and appeared as a mixed population of small spherical (7.8 +/- 1.3 nm) and larger discoidal particles (17.7 +/- 4.9 nm long axis) by electron microscopy. Nondenaturing gradient gel analysis (GGE) of control HDL showed major peaks banding at 7.4, 10.0, 11.1, 12.2, and 14.7 nm. Following 4-h LCAT and 12-h LPDP incubations, HepG2 HDL were mostly spherical by electron microscopy and showed major peaks at 10.1 and 8.1 nm (LCAT) and 10.0 and 8.4 nm (LPDP) by GGE; the particle size distribution was similar to that of plasma HDL. In addition, the chemical composition of HepG2 HDL at these incubation times approximated that of plasma HDL. Molar increases in HDL cholesteryl ester were accompanied by equimolar decreases in phospholipid and unesterified cholesterol. HepG2 low density lipoproteins (LDL) isolated from control samples showed a prominent protein band at 25.6 nm with GGE. Active LPDP or LCAT incubations resulted in the appearance of additional protein bands at 24.6 and 24.1 nm. No morphological changes were observed with electron microscopy. Chemical analysis indicated that the LDL cholesteryl ester formed was insufficient to account for phospholipid lost, suggesting that LCAT phospholipase activity occurred without concomitant cholesterol esterification.     

Uptake of chylomicron remnants causes cholesterol accumulation in cultured human arterial smooth muscle cells

Chylomicron remnants (Sf greater than 100) were prepared by treating human chylomicrons (Sf greater than 400) with human post heparin plasma. Chylomicron remnants recovered after 70-80% of chylomicron triacylglycerol was hydrolyzed, suppressed LDL-receptor activity and increased cell cholesterol esterification to the same extent as did LDL when added to cultured human arterial smooth muscle cells at an equal cholesterol concentration. Cell cholesterol mass increased 36% after incubation with 25 micrograms LDL cholesterol/ml and 35% with 25 micrograms chylomicron-remnant cholesterol/ml. Addition of 30 microM chloroquine plus LDL or chylomicron remnants further increased cholesterol content of cells (74% and 87%, respectively) and caused a significant rise in cell esterified cholesterol (344% and 369%, respectively). Cholesterol content per unit of apolipoprotein B mass of remnants was 2-3-fold higher than that of LDL. Therefore, if lipoprotein particles were added at equivalent apolipoprotein B mass chylomicron remnants increased cell cholesterol content and cholesterol esterification and suppressed LDL receptor activity significantly more than did LDL. This suggests that an additional determinant, presumably apolipoprotein E, is important for receptor recognition of chylomicron remnants. These results may be relevant to the delivery of chylomicron-derived cholesterol to arterial cells proposed as a feature of atherogenesis.     

An enzymatic method for the determination of the initial rate of cholesterol esterification in human plasma.

A method is described for the determination of the initial rate of cholesterol esterification in human plasma, based on the enzymatic determination of free cholesterol in the plasma before and after incubation at 37 degrees C. The cholesterol esterification rate was linear up to 40 minutes. In 18 normal male and 10 normal female subjects the cholesterol esterification rate was 91 +/- 15 (mean +/- SD) and 62 +/- 12 nmoles/hr/ml of plasma, respectively.