Severe HDL deficiency due to novel defects in the ABCA1 transporter.

OBJECTIVES: The objective was the identification and functional characterization of mutations in the ABCA1 gene in four patients with severe HDL deficiency. SUBJECTS: Patients were referred to the clinic because of almost complete HDL deficiency. METHODS: The ABCA1 gene was sequenced directly. The analysis of the ABCA1 protein, ABCA1 mRNA and ABCA1-mediated cholesterol efflux was performed in cultured fibroblasts. Intracellular localization of ABCA1 mutants was investigated in transfected HEK293 cells. RESULTS: Two patients were homozygous for mutations in the coding region of the ABCA1 gene, resulting in an amino acid substitution (p.A1046D) and a truncated protein (p.I74YFsX76). The third patient was homozygous for a splice site mutation in intron 35 (c.4773 + 1g>a), resulting in an in-frame deletion of 25 amino acids (del p.D1567_K1591) in ABCA1. These patients had clinical manifestations of accumulation of cholesterol in the reticulo-endothelial system. The fourth patient, with preclinical atherosclerosis, was a compound heterozygote for two missense mutations (p.R587W/p.W1699C). ABCA1-mediated cholesterol efflux was abolished in fibroblasts from patients with p.A1046D and del p.D1567_K1591 mutants and in fibroblasts homozygous for p.R587W. A reduced ABCA1 protein content was observed in these cells, suggesting an increased intracellular degradation. The mutant p.W1699C was largely retained in the endoplasmic reticulum, when expressed in HEK293 cells. CONCLUSIONS: The homozygotes for mutations which abolish ABCA1 function showed overt signs of involvement of the reticulo-endothelial system. This was not the case in the compound heterozygote for missense mutations, suggesting that this patient retains some residual ABCA1 function that reduces cholesterol accumulation in the reticulo-endothelial system.

Hepatic uptake and processing of free cholesterol from different lipoproteins with and without sodium taurocholate administration. An in vivo study in the rat.

The metabolic fate of [14C]cholesterol carried in the VLDL, LDL, HDL2 and HDL3 lipoprotein fractions has been investigated in bile-fistula rats with jugular vein cannulation. After the depletion of bile salt pool, the labelled lipoprotein fraction was administered either with or without the continuous infusion of sodium taurocholate. The proportion of labelled steroids secreted in the bile within 3 h after the lipoprotein administration was generally higher when the exogenous bile salt was infused. Specifically, the HDL2 fraction induced the secretion of relatively high proportions of labelled steroids, mainly bile salts, either with or without the taurocholate infusion. The other lipoprotein fractions increased the proportion of free cholesterol in steroid bile secretion under taurocholate administration. The label associated to the LDL fraction showed a higher biliary secretion and a more steady clearance from plasma if the exogenous bile salt was not administered. In this same condition, the administration of HDL3 fraction gave the highest values of radioactivity recovered in the liver (mainly as free cholesterol) and a significant increase of cholesterol in the biliary secretion. The present results suggest that each lipoprotein fraction tested may contribute in a peculiar manner to bile salt and cholesterol biliary secretion. Both the expression of apo-E and apo B,E receptors and the levels of circulating bile salts appear to have a role in this process.

Hepatic uptake and metabolism of free cholesterol from different lipoprotein classes. An in vivo study in the rat.

Hepatic metabolism of [14C]cholesterol, vehiculated by LDL, HDL2 and HDL3 lipoprotein particles, has been studied in rats with a permanent biliary drainage. The lipoprotein fractions were infused individually by a jugular vein catheter and bile was collected for 180 min after the administration. At the end of this period, the animals were killed and the blood and livers were collected. The free cholesterol of the HDL2 fraction was secreted into bile, mainly as bile salt, preferentially to that associated with HDL3 and LDL fractions (11.7% vs. 2.3% and 0.3%, respectively). The free cholesterol of the HDL3 fraction, on the other hand, was taken up by liver more quickly and in a higher proportion than that associated with other lipoprotein fractions. The label incorporation in this lipoprotein fraction was secreted earlier and not transformed into bile. The contribution of LDL-vehiculated free cholesterol to bile secretion was small and the hepatic uptake amounted to no more than 12% of the injected label.

The cesium-induced delay in myoblast membrane fusion is accompanied by changes in cellular subfraction lipid composition.

We have recently demonstrated that the delay in myoblast membrane fusion induced by cesium is accompanied by changes in isolated membrane lipids (Santini, M.T., Indovina, P.L., Cantafora, A. and Blotta, I. (1990) Biochim. Biophys. Acta 1023, 298-304). In the present study, we have investigated changes in the lipid profile of total cell homogenates and microsomal membrane fractions during myoblast membrane fusion as well as the effects that addition of cesium may have on these lipid variations in order to try to understand the production and translocation of lipids during this myogenic process. The data presented here indicate that the lipid composition of cell homogenates and microsomes varies in a different manner from isolated plasma membranes during myogenic fusion. In addition, cesium affects, in a different manner, the normally-occurring lipid production and distribution which takes place in each subcellular fraction.

Hepatic uptake and processing of cholesterol and cholesteryl ester from chylomicron remnants: an in vivo study in the rat.

The metabolic fate of cholesterol in chylomicron remnants was studied after intravenous infusion into biliary drained rats. The remnants were radiolabelled with [3H]cholesterol in vivo, so that radioactivity was incorporated into both the unesterified (27% of label) and esterified (73% of label) cholesterol fractions, or with 14C-labelled unesterified cholesterol after exchange in vitro. Blood and bile samples were collected at intervals for 180 min, after which the animals were killed for analysis. The total amount of radioactivity found in the liver (46%) and bile (4.5%) after infusion of [3H] remnants was higher than that found when the label was 14C (33 and 2.7%, respectively). Radioactivity from 14C-labelled unesterified cholesterol was cleared more rapidly from the blood, but the distribution of the label between the lipoprotein fractions VLDL + LDL, HDL2 and HDL3 at the end of the experiment was similar to that found when total [3H]cholesterol was used. In experiments with both types of label, approximately 94% of the total radioactivity secreted into bile was associated with the bile acid, with only about 6% in biliary unesterified cholesterol, and these proportions did not change during 180 min. When the chylomicron remnants were labelled with total [3H]cholesterol the specific radioactivity of the bile acid, taurochenodeoxycholic acid, in the bile was approximately twice that observed when the label was unesterified [14C]cholesterol. The specific radioactivity of unesterified biliary cholesterol was very low in the latter case, but higher and more comparable to that of taurochenodeoxycholic acid in the former. Thus, the metabolic fate of chylomicron remnant cholesterol differs, depending on whether it is in the esterified or unesterified form, suggesting that hepatic cholesterol originating from the two fractions may mix to a different extent with the various intracellular pools. In addition, the experiments with 14C indicate that the behaviour of chylomicron remnant unesterified cholesterol resembles that exhibited by cholesterol in HDL more than that carried in VLDL or LDL.

Transport, utilization and biliary secretion of lysophosphatidylcholine in the rat liver.

The hepatic uptake, transport and utilization of plasma lysophosphatidylcholine (lysoPC) and its contribution to biliary lipid secretion have been investigated in bile-fistula rats. The animals were given a single intravenous dose of sn-1-[1-14C]palmitoyl-lysoPC, under constant intravenous sodium taurocholate infusion (1 mumol/min), and the fate of the label was followed in blood, bile and liver for up to 3 h. The livers were excised at given time points, extracted and/or homogenized to determine the lipid distribution and subcellular location of radioactivity. LysoPC was rapidly cleared from plasma, though a consistent fraction of the label persisted in plasma over the experimental time-period in the form of either lysoPC or PC. Recovery of radioactivity in the liver varied from 15.6% after 5 min to 19.5% after 3 h. Hepatic lysoPC underwent rapid microsomal acylation to form specific PC molecular species (mainly 16:0-20:4 and, to a lesser extent, 16:0-18:2 and 16:0-16:1). Ultrafiltration, dialysis and gel-chromatographic analyses of cytosolic fractions (post 105,000 X g supernatants) indicated that lysoPC is transported to the site of acylation mostly as a macromolecular aggregate with an approx. Mr of 14,400. Small amounts of radioactivity were secreted into bile over 3 h (20% in the form of lysoPC and the remainder as 16:0-18:2 and 16:0-20:4 PC species). Plasma lysoPC, taken up by the liver, is mostly transported by a cytosolic carrier with a molecular weight close to fatty-acid-binding proteins; it then enters a distinct acylation pathway, selective for some polyunsaturated-PC species and does not contribute significantly to biliary secretion, either directly, or through its products.

The cesium-induced delay in myoblast membrane fusion is accompanied by changes in isolated membrane lipids.

We have recently demonstrated that cesium ions delay the sharp decrease in both membrane conductivity and membrane permittivity of chick embryo myoblasts seen at fusion (Santini, M.T., Bonincontro, A., Cametti, C. and Indovina, P.L. (1988) Biochim. Biophys. Acta 945, 56-64). Analysis of the conductivity dispersion data (obtained in the radiowave frequency range) indicated that cesium delays fusion by about 30 h. We suggested that cesium is affecting both active ionic transport by blocking potassium channels as well as interfering with membrane lipid and/or protein charges. In the present study, we have investigated both the possible role of membrane lipids in myoblast fusion and the possible effects of cesium on these lipids. Our data indicate that lipid changes do occur in the isolated myoblast plasma membrane of controls during myogenic differentiation especially prior to fusion and that in cesium cultures these variations do not occur. These variations are in accordance with current membrane fusion theory. Specifically, there is a decrease in bilayer-stabilizing lipids (phosphatidylcholine) and an increase in bilayer-destabilizing ones (phosphatidylethanolamine and phosphatidic acid) and cholesterol during the fusion process. In addition, although slight, during fusion there appears to be a decrease in phosphatidylinositol which is believed to be involved in the inositol phosphate second messenger system. In cesium cultures, in which fusion is greatly delayed, the same lipid changes do not take place and those that are observed seem to reflect the fusion delay.

The lipolysis of chylomicrons derived from different dietary fats by lipoprotein lipase in vitro.

The lipolysis of chylomicrons derived from palm, olive, corn or fish oil (enriched in saturated, monounsaturated, n - 6 polyunsaturated and n - 3 polyunsaturated fatty acids, respectively) by rat post-heparin lipoprotein lipase in vitro was compared by measuring the release of [3H]oleate from their triacylglycerol. Chylomicrons derived from corn oil were lipolysed more rapidly than the other types in the first 20 min of the reaction, but after 120 min the total amount of triacylglycerol hydrolysed was similar with all types of chylomicrons used. The rate of lipolysis of the different types of chylomicrons also showed different dependencies on the substrate concentration. The highest Vmax values were obtained when the chylomicrons were derived from olive and corn oil and the lowest when they were derived from palm oil, while olive oil chylomicrons gave the highest Km and palm oil chylomicrons the lowest. These results indicate that differential metabolism of chylomicrons of different fatty acid composition by lipoprotein lipase may play a part in the differential rates of clearance from the blood of lipid of dietary origin demonstrated in earlier work from our laboratory.

The influence of estrogen on hepatic cholesterol metabolism and biliary lipid secretion in rats fed fish oil.

Both estrogen and dietary n-3 polyunsaturated fatty acids are known to be hypocholesterolemic, but appear to exert their effects by different mechanisms. In this study, the interaction between dietary fish oil (rich in n-3 polyunsaturated fatty acids) and estrogen in the regulation of hepatic cholesterol metabolism and biliary lipid secretion in rats was studied. Rats fed a low fat or a fish oil-supplemented diet for 21 days were injected with 17alpha-ethinyl estradiol (5 mg/kg body weight) or the vehicle only (control rats) once per day for 3 consecutive days. Estrogen-treatment led to a marked reduction in plasma cholesterol levels in fish oil-fed rats, which was greater than that observed with either estrogen or dietary fish oil alone. The expression of mRNA for cholesterol 7alpha-hydroxylase was decreased by estrogen in rats fed a low fat or a fish oil-supplemented diet, while the output of cholesterol (micromol/h/kg b.wt.) in the bile was unchanged in both groups. Cholesterol levels in the liver were increased by estrogen in rats given either diet, but there was a significant shift from cholesterol esterification to cholesteryl ester hydrolysis only in the fish oil-fed animals. Estrogen increased the concentration of cholesterol (micromol/ml) in the bile in rats fed the fish oil, but not the low fat diet. However, the cholesterol saturation index was unaffected. The output and concentration of total bile acid was also unaffected, but changes in the distribution of the individual bile acids were observed with estrogen treatment in both low fat and fish oil-fed groups. These results show that interaction between estrogen-treatment and dietary n-3 polyunsaturated fatty acids causes changes in hepatic cholesterol metabolism and biliary lipid secretion in rats, but does not increase the excretion of cholesterol from the body.

Selective hepatic enrichment of polyunsaturated phosphatidylcholines after intravenous administration of dimethylethanolamine in the rat.

The content of polyunsaturated phosphatidylcholines (PCs) is one of the parameters which regulate membrane functions. Polyunsaturated PCs are preferentially synthesized in the liver by the microsomal enzyme phosphatidylethanolamine N-methyltransferase. The activity of this enzyme may be stimulated in vitro in isolated rat hepatocytes by supplementation with dimethylethanolamine (DME), the polar head group of the precursor of PC along this pathway. The aim of this study was to evaluate in vivo the effect of an intravenous infusion of DME in the rat on the hepatic phospholipid composition. Bile fistula rats were intravenously infused for 15 h with sodium taurocholate (1 mumol/kg per min), with or without the addition of 0.3 mg/kg per min of [14C]DME. The concentration per gram of wet liver of individual phospholipid classes, PC molecular species and of total triacylglycerols, as well as the distribution of radioactivity in liver phospholipids, in rat tissues and body fluids were analyzed. A significant (P less than 0.01) enrichment in PC was found in the liver of DME-infused rats with respect to controls. No differences in the other phospholipid classes were found. DME-infused rats showed a significant (P less than 0.01) decrease in the hepatic concentration of triacylglycerols. At HPLC analysis, the enrichment in PC in DME-infused rats was found to be selectively due to three molecular species (i.e., sn-1 stearoyl/sn-2 arachidonoyl, sn-1 stearoyl/sn-2 linoleoyl, sn-1 stearoyl/sn-2 docosahexanoyl molecular species). In agreement with quantitative data, more than 70% of hepatic radioactivity was recovered in polyunsaturated PC species, with the highest specific activity in the sn-1 stearoyl PCs. The specific activity of hepatic PC approximates that of phosphatidyldimethylethanolamine. This finding together with the effective incorporation of DME in PC suggests that this amino base is methylated after its incorporation into phosphatidyldimethylethanolamine, throughout the stimulation of hepatic N-methyltransferase activity. The selective hepatic enrichment with polyunsaturated PC species after DME infusion may offer a new experimental tool for studying hepatic membrane metabolism.

Development and validation of a quantitative assay for cholesterol crystal growth in human gallbladder bile.

Crystal observation time is a rough estimate of the first microscopic appearance of cholesterol monohydrate crystals in an isotropic bile, and does not provide information on crystal growth kinetics. We have developed a method for quantitating cholesterol crystal growth in gallbladder bile. Crystals were separated from other biliary particles by ultracentrifugation on a discontinuous NaBr gradient, after bile density adjustment to d = 1.060 g/ml. More than 95% of crystals, both of native or synthetic source, floated in the density range 1.045-1.055. This density fraction was collected and crystal mass was measured by photometric turbidity, after calibration with suspensions of different-sized cholesterol crystals. The recovery of crystals added to original bile samples averaged 96.0 +/- 2.8%. Contamination with vesicles, which may potentially interfere with the turbidimetric assay, was excluded by gel-chromatography. The method was sequentially applied, until the 20th day of incubation, to biles obtained at surgery from patients with (A, n = 6) or without cholesterol gallstone (B, n = 4), and from gallstone patients pretreated for 1 week with oral ursodeoxycholic acid (C, n = 5). Crystal growth curves greatly differed, being much steeper in group A and almost flat in patients receiving ursodeoxycholic acid. The mean percent mass of biliary cholesterol in crystalline form at the 20th day was 19.2 +/- 13.5%, 1.2 +/- 0.8% and 2.7 +/- 1.1% in A, B and C, respectively (A vs. B: P = 0.014; A vs. C: P = 0.008). We conclude that the method allows a precise estimate of cholesterol crystal growth and can be usefully applied to human gallbladder biles.

Evaluation in vivo of the differential uptake and processing of high-density lipoprotein unesterified cholesterol and cholesteryl ester in the rat.

The uptake and processing of high-density lipoprotein (HDL) unesterified and esterified cholesterol were compared in vivo in the rat. HDL labelled with 3H in either unesterified cholesterol or cholesteryl ester was administered intravenously, and the clearance of radioactivity from the blood, its distribution in plasma lipoprotein density fractions, uptake by tissues, and appearance in bile were studied at intervals up to 180 min. 3H in HDL unesterified cholesterol was cleared more rapidly from the blood than that in HDL cholesteryl ester, and this difference was mainly due to rapid sequestration of [3H]unesterified cholesterol by the liver, with 58.2% of the administered dose found in this tissue after 10 min, compared to 6.8% of the [3H]cholesteryl ester dose. Non-hepatic tissues took up only a small proportion of the administered label from both HDL unesterified and esterified cholesterol, but on a per gram wet weight basis, the specific uptake of HDL cholesteryl ester in the adrenal glands and the spleen was higher than in the liver, particularly in the first 60 min. The distribution of radioactivity in the plasma lipoprotein density fractions remained constant between 10 and 180 min when [3H]unesterified cholesterol was used, but the proportion of plasma radioactivity from HDL labelled in esterified cholesterol in the very-low-density lipoprotein (VLDL) fraction increased from 0% to 26%, while in HDL there was a shift in the distribution of radioactivity from the most (d 1.125-1.250 g/ml) to the least (d 1.050-1.085 g/ml) dense sub-fractions. A greater percentage of the administered label from HDL unesterified cholesterol (8.8%) than from HDL cholesteryl ester (3.3%) was secreted into bile during 180 min, but the proportions secreted in bile acids and unesterified cholesterol were similar with both labels. These findings indicate that there are significant differences in the uptake and processing of HDL unesterified as compared to esterified cholesterol in the rat in vivo.

The influence of dietary saturated and unsaturated fat on hepatic cholesterol metabolism and the biliary excretion of chylomicron cholesterol in the rat.

The biliary excretion of [3H] cholesterol carried in chylomicrons derived from palm oil (rich in long chain saturated fatty acids), olive oil (rich in monounsaturated fatty acids) or corn oil (rich in n-6 polyunsaturated fatty acids was studied in vivo in rats fed the corresponding oil in the diet for 21 days. The secretion of radioactivity into bile as both bile acids and unesterified cholesterol was significantly slower in the animals fed palm oil as compared to those given olive or corn oil, indicating that dietary saturated fat retards the excretion of cholesterol from the diet as compared to mono- or n-6 polyunsaturated fat. In order to investigate the mechanisms underlying these differences, the influence of the three high fat diets on cholesterol esterification, cholesteryl ester hydrolysis and bile acid synthesis in the liver and on biliary lipid output were also measured. The ratio of cholesterol esterification to cholesteryl ester hydrolysis was markedly raised in the olive and corn oil-fed as compared to palm oil-fed animals. Biliary cholesterol secretion was higher in corn oil-fed rats than in those fed olive or palm oil or a low fat diet, and this was associated with a markedly increased lithogenic index in these animals. The activity of cholesterol 7alpha hydroxylase was higher in the olive and corn oil-fed than in the palm oil-fed animals, although the expression of mRNA for the enzyme was increased only in the olive oil diet group. After 20 h biliary drainage, the rate of bile acid secretion into bile was increased in the rats fed olive and corn oil rather than to palm oil. These findings indicate that feeding rats mono- or n-6 polyunsaturated as compared to saturated fat in the diet promotes the storage of cholesteryl ester in the liver and leads to increased bile acid synthesis, resulting in the more rapid excretion of cholesterol originating from the diet via the bile.

Comparison of the hepatic uptake and processing of cholesterol from chylomicrons of different fatty acid composition in the rat in vivo.

The effect of the fatty acid composition of chylomicrons on the uptake and processing of the cholesterol they carry was investigated in the rat in vivo. Rats kept on a standard low fat pellet diet and tube fed a single dose of palm, olive, corn or fish oil (rich in saturated, n-9 monounsaturated, n-6 polyunsaturated and n-3 polyunsaturated fatty acids, respectively) were used to prepare [3H]cholesterol-labelled chylomicrons of different fatty acid composition. These were then injected intravenously into rats (kept on the standard diet), and the clearance of radioactivity from the blood, distribution in the plasma lipoprotein density fractions, uptake by the liver and appearance in the bile were studied. [3H]Cholesterol from fish and corn oil chylomicrons was cleared from the blood more rapidly than that from palm and olive oil chylomicrons. After 180 min the proportion of the radioactivity present in the plasma in high density lipoprotein (HDL) was less when the chylomicrons were derived from palm oil as compared to any of the other oils. Approx. 40% of the administered label was recovered in the liver after 180 min in all experiments. The percentage of the injected radioactivity secreted into bile during 180 min was significantly higher with corn and fish oil chylomicrons than with palm oil chylomicrons, with chylomicrons from olive oil in an intermediate position, and these differences were most pronounced between 60 and 120 min after administration of the label. These studies clearly demonstrate that the fatty acid composition of chylomicrons has important effects on the hepatic uptake and processing of the cholesterol they carry, with enrichment with polyunsaturated fatty acids leading to an increased rate of uptake and more rapid removal from the body via the bile as compared to enrichment with saturated or monounsaturated fatty acids.

Influence of tauroursodeoxycholic and taurodeoxycholic acids on hepatic metabolism and biliary secretion of phosphatidylcholine in the isolated rat liver.

Studies were carried out using an isolated rat liver system to define: the contribution of exogenous phosphatidylcholine (PC) to biliary phospholipid secretion; and its hepatic metabolism during perfusion of the livers with conjugated bile salts with different hydrophilic/hydrophobic properties. A tracer dose of sn-1-palmitoyl-sn-2-[14C]linoleoylPC was injected as a bolus into the recirculating liver perfusate, under constant infusion of 0.75 mumol/min of tauroursodeoxycholate or taurodeoxycholate. The effects on bile flow, biliary lipid secretion, 14C disappearance from the perfusate and its appearance in bile, as well as hepatic and biliary biotransformation were determined. With both the bile salts, about 40% of the [14C]PC was taken up by the liver from the perfusate over 100 min. During the same period less than 2% of the given radioactivity was secreted into bile. More than 95% of the 14C recovered in bile was located within the identical injected PC molecular species. The biliary secretion of labeled as well as unlabeled PC, however, was significantly higher in livers perfused with taurodeoxycholate than tauroursodeoxycholate, while the reverse was observed with respect to bile flow and total bile salt secretion. The exogenous PC underwent extensive hepatic metabolization which appeared to be influenced by the type of bile salt perfusing the liver. After 2 h perfusion, the liver radioactivity was found, in decreasing order, in PC, triacylglycerol, phosphatidylethanolamine and diacylglycerol. In addition, the specific activity of triacylglycerol was significantly higher in tauroursodeoxycholate than in taurodeoxycholate-perfused livers (P less than 0.025), while the reverse was true for the specific activity of hepatic PC (P less than 0.01). Because taurodeoxycholate and tauroursodeoxycholate showed opposite effects on both biliary lipid secretion and hepatic PC biotransformations, we conclude that the hepatic metabolism of glycerolipids is influenced by the physiochemical properties of bile salts.

Clinical expression of familial hypercholesterolemia in clusters of mutations of the LDL receptor gene that cause a receptor-defective or receptor-negative phenotype.

Seventy-one mutations of the low density lipoprotein (LDL) receptor gene were identified in 282 unrelated Italian familial hypercholesterolemia (FH) heterozygotes. By extending genotype analysis to families of the index cases, we identified 12 mutation clusters and localized them in specific areas of Italy. To evaluate the impact of these mutations on the clinical expression of FH, the clusters were separated into 2 groups: receptor-defective and receptor-negative, according to the LDL receptor defect caused by each mutation. These 2 groups were comparable in terms of the patients' age, sex distribution, body mass index, arterial hypertension, and smoking status. In receptor-negative subjects, LDL cholesterol was higher (+18%) and high density lipoprotein cholesterol lower (-5%) than the values found in receptor-defective subjects. The prevalence of tendon xanthomas and coronary artery disease (CAD) was 2-fold higher in receptor-negative subjects. In patients >30 years of age in both groups, the presence of CAD was related to age, arterial hypertension, previous smoking, and LDL cholesterol level. Independent contributors to CAD in the receptor-defective subjects were male sex, arterial hypertension, and LDL cholesterol level; in the receptor-negative subjects, the first 2 variables were strong predictors of CAD, whereas the LDL cholesterol level had a lower impact than in receptor-defective subjects. Overall, in receptor-negative subjects, the risk of CAD was 2.6-fold that of receptor-defective subjects. Wide interindividual variability in LDL cholesterol levels was found in each cluster. Apolipoprotein E genotype analysis showed a lowering effect of the epsilon2 allele and a raising effect of the epsilon4 allele on the LDL cholesterol level in both groups; however, the apolipoprotein E genotype accounted for only 4% of the variation in LDL cholesterol. Haplotype analysis showed that all families of the major clusters shared the same intragenic haplotype cosegregating with the mutation, thus suggesting the presence of common ancestors.

Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells. NMR and biochemical studies.

RNA-containing vesicles, recovered from the supernatant of high-density cell samples of human colon carcinoma, produce a high-resolution 1H NMR spectrum of lipids characterized by isotropic tumbling; these vesicles contain large amounts of triglycerides and cholesterol esters. Both findings have strict analogies to what is displayed by the proteolipid complexes isolated from the sera of tumor-bearing patients [(1985) Proc. Natl. Acad. Sci. USA 82, 3455-3459; (1986) FEBS Lett. 203, 164-168]. Lipid analysis and enzymatic tests indicate that these vesicles are selected micromaps of plasma membranes, analogous to those that can be recovered from culture media in which tumor cells are grown [(1985) Dev. Biol. 3, 33-57]. Peculiar lipids, an acylated oligopeptide and a modified phospholipid, are also present in the vesicles.

Alterations of lipid composition in Friend leukemia cell tumors in mice treated with tumor necrosis factor-alpha.

Lipid analyses were carried out on transplantable murine Friend leukemia cell tumors, 6 h after intratumoral administration of tumor necrosis factor-alpha (TNF). The levels of the major phospholipid classes were uniformly decreased to about 70% of control values; free fatty acids were increased to about 170%; diacylglycerol was decreased to about 50% and triacylglycerol, the main lipid component, was not significantly altered. These results analysed in the light of concomitant alterations in the levels of phospholipid precursors and catabolites (determined in previous 31P NMR studies) and histological modifications demonstrated that at early stages of TNF-induced inhibition of tumor growth (a) phospholipid catabolism was significantly enhanced; (b) morphological changes were apparently correlated with alterations in the levels of phosphatidylcholine and its catabolic products.

Probucol reduces hepatic cholesterol secretion in hyperlipidemic Yoshida rats.

In this study, perfused livers from Yoshida rats, either on a normal diet or on a diet with 0.3% probucol, were examined. The analysis of liver lipid content and of bile and lipoprotein secretion changes showed that probucol had a relevant effect on liver lipid biosynthesis. In particular, it reduced the production of triacylglycerols and, to a much greater extent that of cholesterol. In addition, probucol reduced plasma cholesterol concentration by decreasing esterified cholesterol in HDL1 and HDL2 fractions. Furthermore, HDL1 composition of both hepatic neosynthetized and circulating particles was strongly modified by probucol. Finally, probucol did not appear to induce significant differences in lipid bile secretion while phospholipid secretion from perfused livers was increased. These facts suggest that the hypolipidemic action of probucol is not mediated by an increase in bile steroid secretion, but rather by a direct reduction in hepatic lipoprotein cholesterol secretion. This secretion induces a modified plasma profile of HDL particles such that these variations are advantageous in terms of reverse cholesterol transport.

The mechanism underlying the hypocholesterolemic effect of chronic fish oil feeding in rats is not due to increased excretion of dietary cholesterol.

The role of the excretion of dietary cholesterol in the hypocholesterolaemic effect of chronic fish oil feeding in rats was investigated. The hepatic uptake and processing of [3H]cholesterol carried in chylomicrons derived from fish oil was studied in vivo in rats fed a low fat diet or a diet supplemented with fish oil for 21 days. In addition, the effects of the fish oil diet on cholesterol esterification, cholesteryl ester hydrolysis, bile acid synthesis and biliary lipid secretion were determined. In rats fed the fish oil as compared to the low fat diet, the uptake of [3H]cholesterol from the blood and its secretion into bile as bile acids was significantly slower, and this was entirely due to a decrease in the bile acid fraction. Biliary bile acid mass secretion was unchanged by fish oil feeding, while biliary cholesterol and phospholipid secretion was increased. No significant differences were observed either in the expression of mRNA for cholesterol 7alpha hydroxylase or the secretion of bile acids into bile after 20 h biliary drainage between the fish oil and low fat diet groups, suggesting that bile acid synthesis is not affected. These results indicate that the access of chylomicron cholesterol to the hepatic substrate pool for bile acid formation is decreased in the fish oil fed rats, and this, together with its slower uptake from the blood, accounts for the retardation of its excretion via the bile. Thus, the hypocholesterolemic effect of dietary fish oil in rats is not due to more rapid metabolism of cholesterol originating from the diet.