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.     

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).     

Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma

Lecithin-cholesterol acyltransferase (LCAT) mass, activity and endogenous cholesterol esterification rate were measured in plasma and apolipoprotein A-I-free (A-I-free) plasma from two normolipidemic and two hyperlipidemic subjects, and from a patient with Tangier disease. A-I was removed from plasma by an anti-A-I immunosorbent. LCAT activity was measured using an exogenous substrate. The plasma LCAT concentration of the four non-Tangier subjects was 4.63 +/- 0.64 micrograms/ml (mean +/- S.D.); means of 26 +/- 7% of total LCAT mass and 22 +/- 11% of plasma LCAT activity were found in their A-I-free plasma. The plasma LCAT concentration of the Tangier subject was 1.49 micrograms/ml. About 95% of LCAT mass and all LCAT activity were found in the A-I-free plasma. Thus, the LCAT mass (1.4 micrograms/ml) and activity (43.1 nmol/h per ml) in Tangier A-I-free plasma were not significantly different from that found in the four non-Tangier A-I-free plasmas (mass = 1.21 +/- 0.44 micrograms/ml; activity: 27.3 +/- 18.4 nmol/h per ml). Although the LCAT activity per unit mass of the enzyme in plasma and A-I-free plasma were comparable (24.9 +/- 2.8 vs. 22.8 +/- 7.8 nmol/h per micrograms LCAT, n = 5), the plasma cholesterol esterification rate of A-I-free plasma from all subjects was lower than that found in plasma (7.5 +/- 2.7 vs. 13.0 +/- 3.8 nmol/h per micrograms LCAT). In conclusion, although A-I-containing lipoproteins are the preferred substrates of LCAT, other LCAT substrates and cofactors are found in A-I-free plasma along with LCAT. Thus, non-A-I-containing particles can serve as physiological substrates for cholesterol esterification mediated by LCAT.     

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.     

Single repeat deletion in ApoA-I blocks cholesterol esterification and results in rapid catabolism of delta6 and wild-type ApoA-I in transgenic mice

The deletion mutation Delta6 apolipoprotein A-I lacks residues 143-164 or repeat 6 in the mature apoA-I protein. In vitro studies show this mutation dramatically reduces the rate of lecithin:cholesterol acyltransferase (LCAT) catalyzed cholesterol esterification. The present study was initiated to investigate the effect of this mutation on in vivo high density lipoprotein (HDL) cholesterol esterification and metabolism. Transgenic mice expressing human Delta6 apoA-I (TgDelta6 +/+) were created and then crossed with apoA-I knockout mice (-/-) to generate mice expressing only human Delta6 apoA-I (TgDelta6 -/-). Human Delta6 apoA-I was associated with homogeneous sized alpha-HDL, when wild-type mouse apoA-I was present (in TgDelta6 +/+ and +/- mice). However, in the absence of endogenous mouse apoA-I, Delta6 apoA-I was found exclusively in cholesterol ester-poor HDL, and lipid-free HDL fractions. This observation coincides with the 6-fold lower cholesterol ester mass in TgDelta6 -/- mouse plasma compared with control. Structural studies show that despite the structural perturbation of a domain extending from repeat 5 to repeat 8 (137-178), Delta6 apoA-I binds to spherical unilamellar vesicles with only 2-fold less binding affinity. In summary, these data show a domain corresponding to apoA-I repeat 6 is responsible for providing an essential conformation for LCAT catalyzed generation of cholesterol esters. Deletion of apoA-I repeat 6 not only blocks normal levels of cholesterol esterification but also exerts a dominant inhibition on the ability of wild-type apoA-I to activate LCAT in vivo.     

Intracellular sterol distribution in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein

The potential role of liver fatty acid binding protein (L-FABP) in modulating cellular sterol distribution was examined in mouse L-cell fibroblasts transfected with cDNA encoding L-FABP. L-cells were chosen because they contain only a small amount of endogenous FABP which does not bind [3H]cholesterol, does not enhance intermembrane sterol transfer, and whose content is unaltered by the expression of L-FABP. Transfected L-cells expressed 0.34% of cytosolic protein as L-FABP. Transfection alone with low expression of L-FABP (0.008% of cytosolic protein) had no effect on any of the parameters tested. Three aspects of cellular sterol transfer were examined. First, cellular sterol uptake, monitored by [3H]cholesterol and the fluorescent sterol, delta-5,7,9(11),22-ergostatetraen-3 beta-ol, was increased 21.5 +/- 2.6% (p less than 0.001) in L-cells expressing L-FABP. This increase was not accounted for by increased sterol esterification in the cells expressing L-FABP. Inhibition of both cholesterol transfer and esterification with 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide from Sandoz abolished the L-FABP related enhancement of both [3H]cholesterol uptake and esterification. Second, plasma membrane transbilayer distribution of sterol, determined by fluorescence methods indicated that the majority of sterol was in the inner leaflet of the plasma membrane. In transfected cells expressing L-FABP, twice as much sterol (28 +/- 4%) was present in the exofacial leaflet of the plasma membrane as compared to that of control cells (15 +/- 2%). Third, expression of L-FABP enhanced sterol transfer from the plasma membrane to microsomes in intact cells. Treatment of [3H]cholesterol or [3H]oleate-loaded cells with sphingomyelinase resulted in increased formation of radiolabeled cholesterol ester, consistent with enhanced microsomal esterification of plasma membrane derived cholesterol. Concomitantly, plasma membrane [3H]cholesterol became less accessible to oxidation by cholesterol oxidase. Sphingomyelinase-stimulated cholesterol esterification was 21 +/- 3% greater in transfected cells. Concomitantly, accessibility of plasma membrane [3H]cholesterol to cholesterol oxidase was decreased 18 +/- 3% in cells expressing L-FABP. These differences are consistent with the ability of L-FABP to influence sterol transport and plasma membrane transbilayer sterol distribution in intact cells.     

Stimulation of cholesterol esterification in hepatic microsomes by lipoproteins from normal and hypercholesterolemic rabbit serum.

Incubation of plasma lipoproteins with rabbit hepatic microsomes enriched the microsomes with free cholesterol and stimulated cholesterol esterification. The rate of cholesterol esterification correlated well (r = 0.96) with the concentration of microsomal free cholesterol. Lipoproteins from normal and hypercholesterolemic serum varied in their propensity to stimulate cholesterol esterification. Among the normal lipoproteins, low density lipoproteins was more stimulatory than either high density lipoproteins or intermediate density lipoproteins. However, the intermediate density lipoproteins fraction from hypercholesterolemic serum was consistently more stimulatory than any of the normal lipoproteins. The augmentation of cholesterol content, when microsomes were exposed to mixed hyperlipidemic lipoproteins, was proportionately much greater than augementation of phospholipid or protein concentration.     

Atherogenic lipoprotein profile in families with and without history of early myocardial infarction

In this study we compared several parameters characterizing differences in the lipoprotein profile between members of families with a positive or negative family history of coronary artery disease (CAD). In addition to regular parameters such as the body mass index (BMI), total plasma cholesterol (TC), low density (LDL-C) and high density (HDL-C) cholesterol and triglycerides (TG) we estimated the fractional esterification rate of cholesterol in apoB lipoprotein-depleted plasma (FER(HDL)) which reflects HDL and LDL particle size distribution. A prevalence of smaller particles for the atherogenic profile of plasma lipoproteins is typical. Log (TG/HDL-C) as a newly established atherogenic index of plasma (AIP) was calculated and correlated with other parameters. The cohort in the study consisted of 29 young (< 54 years old) male survivors of myocardial infarction (MI), their spouses and at least one offspring (MI group; n=116). The control group consisted of 29 apparently healthy men with no family history of premature CAD in three generations, their spouses and at least one offspring (control group; n=124). MI families had significantly higher BMI than the controls, with the exception of spouses. Plasma TC did not significantly differ between MI and the controls. MI spouses had significantly higher TG. Higher LDL-C had MI survivors only, while lower HDL-C had both MI survivors and their spouses compared to the controls. FER(HDL) was significantly higher in all the MI subgroups (probands 25.85+/-1.22, spouses 21.55+/-2.05, their daughters 16.93+/-1.18 and sons 19.05+/-1.33 %/h) compared to their respective controls (men 20.80+/-1.52, spouses 14.70+/-0.98, daughters 13.23+/-0.74, sons 15.7+/-0.76 %/h, p<0.01 to p<0.05). Log(TG/HDL-C) ranged from negative values in control subjects to positive values in MI probands. High correlation between FER(HDL) and Log (TG/HDL-C) (r=0.80, p<0.0001) confirmed close interactions among TG, HDL-C and cholesterol esterification rate. The finding of significantly higher values of FER(HDL) and Log (TG/HDL-C) indicate higher incidence of atherogenic lipoprotein phenotype in members of MI families. The possibility that, in addition to genetic factors, a shared environment likely contributes to the familial aggregation of CAD risk factors is supported by a significant correlation of the FER(HDL) values within spousal pairs (control pairs: r=0.51 p<0.01, MI pairs: r=0.41 p<0.05).     

Relations between particle size of HDL and LDL lipoproteins and cholesterol esterification rate

Particle size of low density (LDL) and high density (HDL) lipoproteins and cholesterol esterification rate in HDL plasma (FER(HDL)) are important independent predictors of coronary artery diseases (CAD). In this study we assessed the interrelations between these indicators and routinely examined plasma lipid parameters and plasma glucose concentrations. In 141 men, healthy volunteers, we examined plasma total cholesterol (TC), triglycerides (TG), HDL and LDL cholesterol (HDL-C, LDL-C) and HDL unesterified cholesterol (HDL-UC). Particle size distribution in HDL and LDL was assessed by gradient gel electrophoresis and FER(HDL) was estimated by radioassay. An effect of particle size and FER(HDL) on atherogenic indexes as the Log(TG/HDL-C) and TC/HDL-C was evaluated. Subjects in the study had plasma concentrations (mean +/- S.D.) of TC 5.2+/-0.9 mmol/l, HDL-C 1.2+/-0.3 mmol/l, TG 2.1+/-1.7 mmol/l, glucose 5+/-0.8 mmol/l. Relative concentration of HDL(2b) was 17.6+/-11.5 % and 14.6+/-11.8 % of HDL(3b,c). The mean diameter of LDL particles was 25.8+/-1.5 nm. The increase in FER(HDL) significantly correlated with the decrease in HDL(2b) and LDL particle size (r = -0.537 and -0.583, respectively, P<0.01) and the increase in HDL(3b,c) (0.473, P<0.01). Strong interrelations among TG and HDL-C or HDL-UC and FER(HDL) and particle size were found, but TC or LDL-C did not have such an effect. Atherogenic indexes Log(TG/HDL-C) and TC/HDL-C correlated with FER(HDL) (0.827 and 0.750, respectively, P<0.0001) and with HDL and LDL particle size.     

Intestinal cholesterol absorption in the chyluria model

Isotopic methods for the measurement of dietary cholesterol absorption were compared with the lymph cholesterol balance procedure in filarial chyluria patients. After a single intravenous injection of radioactive cholesterol, absorption was found to be 746 +/- 136 mg/day by method I, which is based upon the fecal endogenous neutral steroid mass measurement, and 471 +/- 135 mg/day by the simultaneously measured lymph/plasma ratio of cholesterol specific activity (dpm/mg). The corresponding value, determined as the difference between lymph cholesterol transport on a cholesterol-containing diet (1500 mg) and on a cholesterol-free diet, was 622 mg/day. When radioactive cholesterol (1487 mg/day) was fed daily to a second patient, absorption determined by isotopic fecal recovery (353 mg/day) matched that obtained by the lymph balance procedure (326 mg/day). Transudation of plasma cholesterol into the intestinal lymph, estimated by the single intravenous injection of radioactive beta-sitosterol, was independent of both the luminal content of plant sterols and the absorption of dietary cholesterol. The absorption of endogenous cholesterol was calculated by: 1) subtracting the cholesterol originating from plasma (transudation) together with the absorbed dietary cholesterol found in lymph from the total mass of cholesterol transported in lymph, and 2) the lymph balance method, i.e., after interrupting the endogenous cholesterol mucosal uptake by beta-sitosterol feeding (9 g/day) while on a cholesterol-free diet. Endogenous cholesterol was preferentially absorbed compared to dietary cholesterol, but there was no competition for absorption. The major portion of dietary cholesterol found in lymph was esterified, but esterification was not a prerequisite for absorption.     

Hydrogenated fat consumption affects acylation-stimulating protein levels and cholesterol esterification rates in moderately hypercholesterolemic women.

To determine whether hydrogenated fat consumption alters triglyceride metabolism and cholesterol esterification rates, 14 women (65-71 years of age) were provided with each of four diets for 5-week periods according to a randomized cross-over design. The experimental diets contained either soybean oil (SO), low trans squeeze (SQM), medium trans tub (TM), or high trans stick (SM) margarines. Triglyceride uptake by adipose tissue was determined by measuring plasma acylation-stimulating protein (ASP), FFA, glucose, and insulin levels, while rates of transfer and esterification rate of newly synthesized cholesterol (ER) were derived by using plasma CETP levels and the deuterium incorporation methodology. Plasma ASP levels were lowest (P < 0.05) in subjects on the SM diet (33.4 +/- 12.7 nM) compared with the SO (48.7 +/- 17.0 nM) and SQM (50.7 +/- 15.7 nM) diets. Conversely, FFA were highest (P < 0.05) on the SM diet (0.86 +/- 0.45 mM) relative to all the other diets. No differences were observed in plasma glucose and insulin levels among diets. A trend toward higher CETP levels after consumption of the SM diet was observed. However, the ER was lowest (P < 0.05) after the SM (0.111 +/- 0.062 g x day(-1)) diet and highest after consumption of the SQM (0.216 +/- 0.123 g x day(-1)) diet. In addition, ASP levels were negatively correlated with FFA (r = -0.63, P < 0.05), LDL cholesterol (r = -0.56, P < 0.05), and TG (r = -0.41, P < 0.05), whereas FFA was positively correlated with apolipoprotein B-containing lipoproteins (r = 0.58 and 0.47, for VLDL and LDL cholesterol, P < 0.05), and negatively correlated with HDL cholesterol (r = -0.51, P < 0.05). The ER was found to positively correlate with HDL cholesterol and HDL2 subfraction (r = 0.53 and 0.45, respectively, P < 0.05). Taken together, these data demonstrate that the alterations in circulating lipid levels, commonly observed with consumption of hydrogenated fat-rich diets, can be explained in part by changes in ASP activity as well as newly synthesized cholesterol ER.     

Cholesterol esters as growth regulators of lymphocytic leukaemia cells

Objective: Alterations in plasma lipid profile and in intracellular cholesterol homoeostasis have been described in various malignancies; however, significance of these alterations, if any, in cancer biology is not clear. The aim of the present study was to investigate a possible correlation between alterations in cholesterol metabolism and expansion of leukaemia cell numbers. Materials and methods: Lipid profiles in plasma and in primary leukaemia cells isolated from patients with acute or chronic lymphocytic leukaemia (ALL and CLL) were studied. Results and conclusions: Decreased levels of HDL‐C were observed in plasma of leukaemic patients, levels of total cholesterol, LDL‐C, triglycerides and phospholipids were unchanged or only slightly increased. As compared to normal lymphocytes, freshly isolated leukaemic cells showed increased levels of cholesterol esters and reduction in free cholesterol. Growth stimulation of ALL and CLL cells with phytohemagglutinin led to further increase in levels of cholesterol esters. Conversely, treatment with an inhibitor of cell proliferation such as the mTOR inhibitor, RAD, caused decline in population growth rate of leukaemia cells, which was preceded by sharp reduction in rate of cholesterol esterification. On the other hand, exposure of leukaemic cells to two inhibitors of cholesterol esterification, progesterone and SaH 58‐035, caused 60% reduction in their proliferation rate. In addition to demonstrating tight correlation between cell number expansion and cholesterol esterification in leukaemic cells, these results suggest that pathways that control cholesterol esterification might represent a promising targets for novel anticancer strategies.     

Role of insulin in regulation of high density lipoprotein metabolism

The effect of alloxan-induced insulin deficiency on high density lipoprotein (HDL) metabolism was studied in rabbits. Rabbits with alloxan-induced diabetes had significantly higher (P less than 0.001, mean +/- SEM) plasma concentrations of glucose (541 +/- 13 vs. 130 +/- 2 mg/dl), triglyceride (2851 +/- 332 vs. 101 +/- 10 mg/dl), and total plasma cholesterol (228 +/- 55 vs. 42 +/- 4 mg/dl) than did normal control rabbits. However, diabetic rabbits had lower plasma HDL-cholesterol (7.2 +/- 1 vs. 51.3 +/- 1.3 mg/dl, P less than 0.001) and HDL apoA-I (38.3 +/- 6.0 vs. 87.2 +/- 4.3 mg/dl, P less than 0.001) concentrations. HDL kinetics were compared in diabetic and normal rabbits, using either 125I-labeled HDL or HDL labeled with 125I-labeled apoA-I, and it was demonstrated that HDL fractional catabolic rate (FCR) was slower and residence time was longer in the diabetic rabbits when either tracer was used. The slow FCR and the low apoA-I pool size led to reduced apoA-I/HDL synthetic rate in diabetic rabbits (0.97 +/- 0.11 vs. 0.34 +/- 0.07 mg per kg per hr). Thus, the reduced plasma HDL-cholesterol concentrations seen in rabbits with alloxan-induced insulin deficiency was associated with a lower total apoA-I/HDL synthetic rate. Since insulin treatment restored to normal all of the changes in plasma lipoprotein concentration and kinetics seen in diabetic rabbits, it is unlikely that the phenomena observed were secondary to a nonspecific toxic effect of alloxan. These data strongly support the view that insulin plays an important role in regulation of HDL metabolism.     

Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts

The purpose of this study was to determine the capacity of Niemann-Pick type C (NPC) fibroblasts to transport cholesterol from the cell surface to intracellular membranes. This is relevant in light of the observations that NPC cells display a sluggish metabolism of LDL-derived cholesterol, a phenomenon which could be explained by a defective intracellular transport of cholesterol. Treatment of NPC cells for 4 h with 0.1 mg/ml of LDL failed to increase the incorporation of [14C]oleic acid into cholesterol [14C]oleate, an observation consistent with previous reports on this cell type (Pentchev et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8247). Normal fibroblasts, however, displayed the classical upregulation (6-fold over control) of the endogenous esterification reaction in response to LDL exposure. Incubation of normal or NPC fibroblasts with sphingomyelinase (100 mU/ml; Staphylococcus aureus) led to a rapid and marked increase (9- and 10-fold for normal and NPC fibroblasts, respectively, after 4 h) in the esterification of plasma-membrane-derived [3H]cholesterol suggesting that sphingomyelin degradation forced a net transfer of cholesterol from the cell surface to the endoplasmic reticulum. The similar response in normal and mutant fibroblasts to the degradation of sphingomyelin suggests that plasma membrane cholesterol can be transported into the substrate pool of ACAT to about the same extent in these two cell types. Degradation of cell sphingomyelin in NPC fibroblasts also resulted in the movement of 20-25% of the cellular cholesterol from a cholesterol oxidase susceptible pool into oxidase-resistant pools, implying that a substantial amount of plasma membrane cholesterol was internalized after sphingomyelin degradation. This cholesterol internalization was not accompanied by an increased rate of membrane internalization, as measured by [3H]sucrose uptake. Although NPC cells showed a relative accumulation of unesterified cholesterol and a sluggish esterification of LDL-derived cholesterol when exposed to LDL, these cells responded like normal fibroblasts with regard to their capacity to transport cholesterol from the cell surface into intracellular sites in response to sphingomyelin degradation. It therefore appears that NPC cells, in contrast to the impaired intracellular movement of lipoprotein-derived cholesterol, do not display a general impairment of cholesterol transport between the cell surface and the intracellular regulatory pool of cholesterol.     

Hepatic lipase and the clearing reaction: studies in euthyroid and hypothyroid subjects

Eight patients with primary hypothyroidism were compared to eleven euthyroid subjects with regard to the effects of a single i.v. dose of heparin on plasma lipoprotein concentrations (the "clearing reaction"). The hypothyroid patients were moderately hypercholesterolemic but had normal plasma triglyceride levels. Maximal activities of hepatic lipase (HL) and lipoprotein lipase (LPL) were lower in the hypothyroid than in the normal subjects. The hypothyroid patients demonstrated a significant decrease in total plasma cholesterol levels after heparin injection (from 8.36 +/- 0.70 mmol/l to 7.55 +/- 0.62 mmol/l, P less than 0.02). The maximal activity of HL after heparin was significantly correlated to the decrease in plasma cholesterol levels (P less than 0.05) and in LDL-cholesterol levels (P less than 0.01). The euthyroid subjects demonstrated a smaller decrease in total plasma cholesterol concentrations (from 5.53 +/- 0.31 to 5.08 +/- 0.28 mmol/l, P less than 0.05). In this group, the fall in cholesterol levels was not correlated to maximal HL activity. The reduction in plasma triglyceride levels after heparin was similar and significant (P less than 0.01) in both groups. These data support the view that decreased activity of HL contributes to the dyslipoproteinemia seen in hypothyroidism. They are also in accordance with the notion that HL is involved in the elimination of cholesterol from plasma.     

Role of plasma lecithin:cholesterol acyltransferase in the metabolism of high density lipoproteins

The role of the plasma lecithin:cholesterol acyltransferase reaction in the esterification of the cholesterol of human and baboon plasma high density lipoproteins has been studied. Human plasma was incubated in vitro, and the initial rate of cholesterol esterification in lipoprotein fractions obtained by chromatography on hydroxylapatite was determined. The rate of esterification was greater in the high density lipoprotein fraction than in the low density lipoprotein fraction. High density lipoproteins from human and baboon plasma were filtered through columns of Sephadex G 200, and the relative concentrations in the effluent of key lipids involved in the acyltransferase reaction were determined. The ratio of esterified to unesterified cholesterol varied across the lipoprotein peak obtained from either type of plasma. The relative concentration of lecithin compared to sphingomyelin also varied across the peaks obtained with human high density lipoproteins. When human or baboon plasma was incubated with cholesterol-(14)C and the high density lipoproteins were filtered through Sephadex, the specific activity of the esterified cholesterol varied across the lipoprotein peak. Similar results were obtained when plasma esterified cholesterol was labeled in vivo by the injection of labeled mevalonate into baboons. The data suggest that the acyltransferase reaction is the major source of the esterified cholesterol of the high density lipoproteins.     

Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae

The Niemann-Pick C1 (NPC1) protein regulates cholesterol transport from late endosomes-lysosomes to other intracellular compartments. In this article, cholesterol transport to caveolin-1 and caveolin-2 containing compartments, such as the trans-Golgi network (TGN) and plasma membrane caveolae, was examined in normal (NPC+/+), NPC heterozygous (NPC+/-), and NPC homozygous (NPC-/-) human fibroblasts. The expression and distribution of NPC1 in each cell type were similar, and characterized by a finely dispersed, granular staining pattern. The expression of caveolin-1 and caveolin-2 was increased in NPC+/- and NPC-/- fibroblasts, although the distribution in each cell type was similar and characterized by predominant staining of the TGN and plasma membrane. The TGN in NPC+/+ fibroblasts was relatively cholesterol-enriched, whereas the TGN in NPC+/- and NPC-/- fibroblasts was partially or completely cholesterol-deficient, respectively. Consistent with studies demonstrating the transport of cholesterol from the TGN to plasma membrane caveolae, the concentration of cholesterol in plasma membrane caveolae isolated from NPC+/- and NPC-/- fibroblasts was significantly decreased, even though the total concentration of plasma membrane cholesterol in each cell type was similar.These studies demonstrate that NPC1 regulates cholesterol transport to caveolin-1 and caveolin-2 containing compartments such as the TGN and plasma membrane caveolae.     

Naringenin 7-O-cetyl ether as inhibitor of HMG-CoA reductase and modulator of plasma and hepatic lipids in high cholesterol-fed rats

Numerous studies in vitro have shown a close relationship between the chemical structure and biologic activity of flavonoids, whereby their basic structure is modified to increase or decrease their biologic activity. The effects of naringenin (1) and its synthetic derivative, naringenin 7-O-cetyl ether (2), on the lipid profile, the cholesterol-regulating enzyme activity and the excretion of sterol were compared in rats fed a high-cholesterol (1% wt/wt) diet. Either 1 or 2 was supplemented with a high-cholesterol diet for 6 weeks at a dose of 0.073 mmol/100g diet. The supplementation of 1 or 2 significantly lowered the levels (mean+/-SE) of the plasma total cholesterol (4.93+/-0.19 and 4.75+/-0.16 mmol/L vs 5.87+/-0.36 mmol/L, p<0.05) and hepatic triglyceride (0.12+/-0.01 and 0.11+/-0.01 mmol/g vs 0.18+/-0.01 mmol/g, p<0.05) and cholesterol (0.23+/-0.01 and 0.21+/-0.01 mmol/g vs 0.31+/-0.01 mmol/g, p<0.05) compared to those of the control. The compound 1 or 2 supplementation appeared to decrease the excretion of neutral sterols. The plasma HDL-cholesterol concentration and ratio of HDL to total cholesterol were significantly higher in 1 and 2 groups than in control group. Although the biological effect of 2 on inhibiting hepatic HMG-CoA reductase and ACAT activities was only significant compared to the control group, both compounds exhibited a significant hypocholesterolemic effect in rats fed a high-cholesterol diet. The results suggest that cholesterol biosynthesis and esterification were concomitantly reduced by 2, as indicated by the decreased HMG-CoA reductase and ACAT activities.     

Esterification of cholesterol in fish plasma: studies on the cholesterol esterifying enzyme in plasma of char (Salmo alpinus L.).

1. Plasma of sea char (Salmo alpinus L.) has the ability to esterify cholesterol in vitro. Heat inactivation of the plasma totally inhibited the esterification. 2. Arrhenius plot of the esterification of [3H]cholesterol was linear in the temperature range 5-35 degrees C. 3. Cholesterol esterification in sea char plasma could not be reversibly inhibited by sulphydryl-blocking agents. Total equilibration of the added [3H]cholesterol and the endogenous cholesterol could therefore not be obtained. 4. Comparison of esterification of endogenous and labelled cholesterol revealed that the isotopic method was valid only in qualitative measurements.     

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)