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PubMed1995

Effect of apolipoprotein A-I deficiency on lecithin:cholesterol acyltransferase activation in mouse plasma.

Plasma cholesteryl ester (CE) synthesis by lecithin cholesterol acyltransferase (LCAT) is activated by apolipoprotein (apo)A-I. We studied the effect of plasma apoA-I concentration on LCAT activation, using normal, heterozygous or homozygous apoA-I-deficient mice made by gene targeting. Plasma esterified cholesterol concentrations of mice fed chow diets were ordered (mean +/- SEM): 105 +/- 7 (normal) > 70 +/- 5 (heterozygotes) > 26 +/- 2 (homozygotes) mg/dl. Plasma free cholesterol concentrations were similar among the three genotypes. Endogenous LCAT activity, measured as the decrease in plasma free cholesterol after a 1 h incubation at 37 degrees C, was ordered: 44 +/- 3 (normal) > 21 +/- 2 (heterozygotes) > 5 +/- 1 (homozygotes) nmol CE formed/h per ml plasma. Using a recombinant exogenous substrate consisting of egg yolk phospholipid, [14C]cholesterol, and apoA-I, CE formation of normals and heterozygotes was similar (27.4 +/- 0.6 and 28.8 +/- 1.3 nmol/h per ml plasma, respectively), but was significantly less for homozygotes (19.2 +/- 1.7 nmol/h per ml plasma). However, using a small unilamellar vesicle substrate particle containing phospholipid and [14C]cholesterol, CE formation was ordered: 1.6 +/- 0.1 (normal) = 1.6 +/- 0.1 (heterozygotes) > 0.6 +/- 0.1 (homozygotes) nmol/h per ml plasma; addition of apoA-I to the plasma of homozygous animals restored CE formation to normal levels (1.6 +/- 0.1). CE fatty acid analysis demonstrated that plasma from homozygous mice contained significantly more saturated and monounsaturated and fewer polyunsaturated fatty acids compared to normal and heterozygous mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

[Lecithin: cholesterol acyltransferase].

Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme that catalyzes esterification of plasma lipoprotein unesterified cholesterol, and plays a central role in maintaining reverse cholesterol transport through action with high density lipoprotein (HDL) and Cholesterol ester transfer protein (CETP). Cloned cDNA sequence of LCAT, consisted of about 1400 base pair which codes 416 amino acids, revealed high content of alpha-helix and beta-sheet, identity of amino acid sequence with various lipases, and putative structure of catalytic sites and mechanism of enzymatic action is proposed. Close relationship between location of gene mutation and severity of clinical and biochemical characteristics revealed by gene analysis in patients of LCAT deficiency and Fish eye disease is summarized.

Amino Acid Sequence↗
PubMed1994

[An intravenously injectable emulsified iodinated oil contrast agent for liver CT: experimental study of lipiodol emulsion emulsified by lecithin (LEL38) in rabbits].

LEL38 (lipiodol emulsion emulsified by lecithin 38 mgI/ml, mean diameter 200 nm) is a new intravenously injectable oil contrast agent for liver CT. The aim of this report was to evaluate its ability to enhance contrast in the liver of 46 rabbits with regard to the correlation of density with time at an injected dose of 76 mgI/kg (before to 120 minutes after), the correlation of density with dose (0-760 mgI/kg) and the detectability of liver mass. The time-density correlation of LEL38 in the liver, that is, peak density, was achieved after 30 minutes, and it was elevated to 20.9 H. U. Thereafter, it decreased slowly. In the blood vessels, it reached a sharp peak after immediately being elevated to 14. 7 H. U. There after detectability decreased quickly. The maximum difference in density between liver and blood vessels was 34 H. U. after 60 minutes. The dose-density correlation in the liver and blood vessels was linear. Tumors were detected as clear areas of low density. The minimal detectability was about 3 mm. LEL38 may be an effective contrast agent for screening CT studies of liver disease.

Animals↗
PubMed1994

Lecithinized superoxide dismutase enhances its pharmacologic potency by increasing its cell membrane affinity.

We performed the present study to clarify whether lecithinized superoxide dismutase (PC-SOD) enhanced its pharmacologic potency by increasing its cell membrane affinity. PC-SOD, in which 4 molecules of a phosphatidylcholine (PC) derivative were covalently bound to each dimer of recombinant human CuZn-SOD (rhCuZn-SOD), was shown to have a high membrane affinity using a laser confocal imaging technique. PC-SOD efficiently scavenged superoxide anion (O2-) produced by phorbol myristate acetate (PMA)-stimulated human neutrophils (IC50 0.60 U/ml), and it exerted a dose-dependent scavenging effect (IC50 1.27 U/ml) even when the neutrophils were washed after incubation with PC-SOD. In contrast, neither unmodified SOD nor polyethylene glycol-bound SOD (PEG-SOD) showed a scavenging effect for washed neutrophils, even at a high concentration (100 U/ml). PC-SOD also showed a strong protective effect against human vascular endothelial cell damage caused by O2- generated by stimulated neutrophils, and PC-SOD was approximately 100-fold more potent than unmodified SOD (in vitro IC50 100 U/ml for PC-SOD and > 10,000 U/ml for unmodified SOD). Moreover, PC-SOD (50,000 U/kg) had an inhibitory effect on ischemia-reperfusion paw edema in mice, whereas neither unmodified SOD nor PEG-SOD had any effect. These results suggest that PC-SOD (designed to target for cell membranes) exerted a far higher pharmacologic activity by increasing cell membrane affinity than unmodified SOD and may be potentially useful for various clinical applications.

Animals↗
PubMed1994

Binding of lecithin:cholesterol acyltransferase to reconstituted high density lipoproteins is affected by their lipid but not apolipoprotein composition.

The reaction of lecithin:cholesterol acyltransferase (LCAT) with high density lipoproteins (HDL) is of critical importance in reverse cholesterol transport. We studied the relationship between LCAT binding and HDL composition using two assays to determine LCAT binding affinity for discoidal reconstituted HDL (rHDL). We prepared rHDL with egg phosphatidylcholine (egg-PC), cholesterol, and either apolipoprotein (apo) A-I or apoA-II. The rHDL, identical in lipid content, had sizes of 96 and 100 A, respectively. Binding constants (Kd) determined by the activity-inhibition method were 2.2 +/- 0.3 x 10(-7) and 1.1 +/- 0.3 x 10(-16) M, whereas those determined by a solid-phase method were 3.3 +/- 0.9 x 10(-7) and 3.7 +/- 0.9 x 10(-7) M for apoA-I and apoA-II rHDL, respectively. The stoichiometry was 1 LCAT bound/rHDL. The appVmax/appKm for apoA-I was 80-fold higher than for apoA-II rHDL. The large difference between LCAT-binding constants and enzymatic activity measurements for the two particles suggests that LCAT binding and activation by apolipoproteins are independent events. To determine the effects of phospholipid headgroup on LCAT binding affinity, we tested rHDL containing up to 16 mol % of egg phosphatidylethanolamine (egg-PE), egg phosphatidic acid (egg-PA), or bovine phosphatidylserine (PS). Kd was unchanged for PS rHDL, but increased 6-fold with increasing PE content. AppVmax increased with PE content, but decreased with PS or PA relative to egg-PC controls. AppKm increased in PE rHDL, but remained unchanged in rHDL with PA or PS. Fluorescence characterization of the lipid domains of rHDL shows small differences in the polarity of the headgroup region of PE rHDL. Thus, LCAT binding is influenced by the lipid, but not the protein composition of rHDL. AppVmax values reflect active site preferences, while appKm values reflect interfacial binding affinity.

Apolipoproteins↗
PubMed1994

High density lipoproteins with differing apolipoproteins: relationships to postprandial lipemia, cholesteryl ester transfer protein, and activities of lipoprotein lipase, hepatic lipase, and lecithin: cholesterol acyltransferase.

To gain insight into metabolic determinants of high density lipoproteins (HDL) containing apolipoproteins A-I and A-II (LpA-I/A-II) and those containing A-I, but devoid of A-II (LpA-I), the plasma concentration of LpA-I and LpA-I/A-II within the HDL2 and HDL3 density spectrum was measured in 14 normolipidemic male subjects on a standardized diet. Apolipoprotein plasma concentrations of HDL subspecies were compared with the magnitude of postprandial lipemia, activities of lipoprotein lipase and hepatic lipase in postheparin plasma, plasma lecithin:cholesterol acyltransferase (LCAT) activity, and cholesteryl ester transfer protein (CETP) mass. Plasma levels of LpA-I/A-II were 2.5 times higher than levels of LpA-I (123 +/- 20 vs. 48.3 +/- 22.1 mg protein/dl) and the partition of LpA-I and LpA-I/A-II between HDL2 and HDL3 differed in that the proportion of LpA-I associated with HDL2 was greater than that of LpA-I/A-II (23 +/- 19 vs. 6 +/- 6%, P < 0.002). With increasing levels of HDL2, the proportion of LpA-I in HDL2 increased (P < 0.002). Furthermore, levels of LpA-I and LpA-I/A-II were strongly correlated within the HDL2 but not within the HDL3 density region. Plasma levels of LpA-I, but not LpA-I/A-II, were inversely correlated with the magnitude of postprandial lipemia. However, activities of lipoprotein lipase and hepatic lipase tended to show stronger associations with the partition of LpA-I/A-II between HDL2 and HDL3 than with that of LpA-I. Within the HDL3, but not the HDL2 density spectrum, LpA-I/A-II exhibited a positive association with plasma LCAT activity, while LpA-I displayed an inverse association with plasma CETP mass. These results are consistent with differences in substrate properties of LpA-I and LpA-I/A-II for lipoprotein modifying enzymes and imply different, but overlapping metabolic pathways of LpA-I and LpA-I/A-II.

Adult↗
PubMed1993

A comparison of the accuracy of the TDx-FLM assay, lecithin-sphingomyelin ratio, and phosphatidylglycerol in the prediction of neonatal respiratory distress syndrome.

OBJECTIVE: To ascertain whether the TDx-FLM assay is a better predictor of neonatal respiratory distress syndrome (RDS) than the lecithin-sphingomyelin ratio (L/S) and phosphatidylglycerol (PG). METHODS: Amniotic fluid samples of 140 women requiring assessment of fetal lung maturity and delivering within 72 hours of amniocentesis were analyzed by the three methods. The accuracy in predicting RDS was assessed by conventional statistical techniques. RESULTS: The TDx-FLM test had a better sensitivity (89.6 versus 48.2%; relative risk [RR] 3.9, 95% confidence interval [CI] 1.35-11.23, P = .001) and negative predictive value (96.4 versus 86.8%; RR 0.36, 95% CI 0.12-1.04, P = .02) than the L/S. However, the L/S had a better specificity (89.1 versus 73.8%; RR 0.64, 95% CI 0.49-0.82, P = .005) than the TDx-FLM assay. The PG test had a predictive ability similar to that of the TDx-FLM assay. However, there was no significant difference in the overall accuracy of the three tests, and the areas under the receiver-operating characteristic curves for the L/S and TDx-FLM (0.80 and 0.87) were similar. CONCLUSIONS: The TDx-FLM assay performs similarly to the L/S and PG tests in the prediction of RDS and is quantitative, rapid, and reproducible.

Albumins↗
PubMedDate not supplied

[Fatty acid composition of lecithin and cholesterol esters of high density lipoproteins from blood of humans, some mammals, and fish].

Fatty acid composition of lecithin and cholesterol esters was studied in high density lipoproteins obtained from blood serum of 20 male volunteers, 10 pigs, 10 rats and 30 burbots (Lota lota); the study was carried out using isolation (precipitation) of the lipoproteins, thin-layer chromatography of lipoproteins and gas liquid chromatography of fatty acids. The findings suggest that the fatty acid composition of the preparations studied may serve as a more exact index of lipid metabolism than the serum levels of individual lipoproteins.

Adult↗
PubMed1993

[Analysis of immunosuppressive activity of lecithin-bound iodine (LBI) on antigen-induced IL2 responsiveness in peripheral blood lymphocytes from patients with allergic diseases].

The effect of lecithin-bound iodine (LBI) on antigen-induced IL2 responsiveness, useful in in vitro test for the detection of etiological antigen and disease activity in allergic diseases, was investigated. In peripheral blood mononuclear cells (PBMC) from asthmatic children, LBI suppressed Dermatophagoides farinae (Df)-induced IL2 responsiveness in a dose dependent manner, in a range of 0.01 to 100 micrograms/ml. Similarly, LBI also suppressed ovalbumin (OVA)-induced IL2 responsiveness in PBMC from patients with atopic dermatitis, sensitized with OVA, in concentration of 0.1 to 100 micrograms/ml. Moreover, purified protein derivative (PPD)-induced IL2 responsiveness was equally inhibited by LBI, at doses of between 0.1 and 100 micrograms/ml. However, LBI could not overcome the responses induced by concanavalin A (Con A) until it reached unphysiological concentrations of as high as 100 micrograms/ml. The fact that antigen-induced IL2 responsiveness was suppressed by LBI at concentrations comparable to blood levels of LBI in individuals medicated suggests that LBI is a weak immunosuppressant. IL2-responding T cells were more sensitive to LBI than antigen-presenting cells in Df-induced IL2 responsiveness, although LBI suppressed both cell populations. Taken together, LBI could work as a weak immunosuppressant to inhibit lymphocyte responses to allergen and to improve clinical status in allergic diseases, including bronchial asthma caused by Df and atopic dermatitis provoked by OVA.

Antigens↗
PubMed1993

Rapid determination by centrifugal ultrafiltration of inter-mixed micellar/vesicular (non-lecithin-associated) bile salt concentrations in model bile: influence of Donnan equilibrium effects.

We have developed the technique of rapid (< 2 h) centrifugal ultrafiltration to measure the inter-mixed micellar/vesicular (non-lecithin-associated) bile salt (BS) concentrations (IMC) of individual BS in model biles. This methodology uses a centrifugal concentrator with a reinforced membrane, through which a small fraction (< 15% total volume) of model biles was ultrafiltered by low centrifugal forces (1500 g 5-60 min). Total and individual BS concentrations in the filtrate were measured by high performance liquid chromatography. However, non-filterable anions, in this case BS/lecithin/cholesterol mixed micelles and unilamellar vesicles, induce an asymmetric distribution of ions across the membrane. Therefore, BS concentrations in the filtrate exceeded the true IMC, which was estimated taking into account Donnan equilibrium effects. To confirm the hypothesis that a correction for Donnan forces was necessary, distributions of BS and another filterable anion, chloride, were measured in systems containing the non-filterable polyanion dextran sulfate as an analogue for non-filterable polyanionic mixed micelles and vesicles. An asymmetric distribution of the monovalent anions chloride and BS monomers as well as polyvalent simple BS micelles was indeed present during centrifugal ultrafiltration. This new methodology was validated by comparing IMC values with those obtained by modified equilibrium dialysis also corrected for Donnan equilibrium effects (Donovan, J.M., et al. 1991. J. Lipid Res. 32: 1501-1512). Centrifugal ultrafiltration, which utilizes < 1 ml of bile, determines the composition in the IMC necessary to separate micelles and vesicles of native biles by techniques that involve dilution of bile such as gel filtration chromatography.

Bile↗
PubMed1993

Radiation inactivation analysis of acyl-CoA:retinol acyltransferase and lecithin:retinol acyltransferase in rat liver.

Microsomes from liver and several other tissues esterify retinol through both fatty acyl-CoA-dependent and -independent reactions. Two activities, acyl-CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT) activities, have been characterized enzymatically but neither has yet been purified and characterized biochemically. We have used the method of radiation inactivation to determine the target sizes of ARAT and LRAT in intact microsomal membranes from rat liver. After exposure of frozen liver microsomes to ionizing radiation, the activity of ARAT decayed exponentially yielding a target size of 73 +/- 18 kDa (mean +/- SD, n = 6). The activity of LRAT was assayed both by monitoring the esterification of retinol bound to the cellular retinol-binding protein (CRBP) and of solvent-dispersed retinol. With both assays a single exponential was observed with radiation doses of 9 to 150 Mrads. The slopes obtained with both LRAT assays were similar, yielding target sizes of 52 +/- 10 kDa (n = 10) for the LRAT assay with CRBP-retinol and 56 +/- 7 kDa (n = 6) for the LRAT assay with dispersed retinol. These target sizes did not differ from each other but were significantly smaller than that of ARAT. These data provide the first physical evidence of the independent entities which catalyze the ARAT and LRAT reactions of liver microsomes.

Acyltransferases↗
PubMed1993

Expression and characterization of recombinant human lecithin:cholesterol acyltransferase.

We have established a baby hamster kidney (BHK) cell line that constitutively expresses significant quantities of human recombinant lecithin:cholesterol acyltransferase (rLCAT). LCAT cDNA was cloned into a mammalian expression vector containing the metallothionein promoter and the dihydrofolate reductase gene. After transfection, the BHK cells were treated with 500 microM methotrexate for 2 weeks to select the successfully transfected cells. Surviving colonies were subcloned and high level secretors were identified by measurement of LCAT activity and mass in the culture medium. The attachment of transfected cells to microcarrier beads enabled the efficient production of large quantities of rLCAT in a serum-free medium. After a single-step chromatography procedure, the rLCAT was purified to homogeneity with yields exceeding 1 mg of rLCAT per 100 ml of culture medium. The molecular weight of rLCAT (approximately 66,000) was identical to that of purified human plasma LCAT on SDS polyacrylamide electrophoresis. The rLCAT was activated by apolipoprotein A-I and had an average specific activity that was similar to purified plasma LCAT. After selective deglycosylation with either neuraminidase or N-glycanase, rLCAT and plasma LCAT had identical molecular weights. The simplification of the production and purification of rLCAT reported here will enable a more in depth analysis of the structure and function of this enzyme.

Animals↗
PubMed1993

Role of sphingomyelin in the regulation of cholesterol esterification in the plasma lipoproteins. Inhibition of lecithin-cholesterol acyltransferase reaction.

In order to determine whether sphingomyelin (SPH) affects the rate of cholesterol esterification by plasma lecithin-cholesterol acyltransferase (LCAT), we studied the effects of its incorporation in to defined proteoliposome substrates containing phosphatidyl choline (PC), unesterified cholesterol, and apoprotein A-I, on the activity of purified LCAT. Cholesterol esterification was inhibited by up to 90% in the presence of SPH, and this inhibition was reversed by treatment with bacterial sphingomyelinase. The inhibition could be overcome by increasing the concentration of PC, but not unesterified cholesterol or apoprotein A-I, in the substrate. The effect of SPH was not related to the alterations in the size of the substrate particle and was not dependent on the type of acyl donor or apoprotein activator employed. The lysolecithin acyltransferase and phospholipase reactions carried out by LCAT were also inhibited by SPH. Kinetic studies suggested that: 1) LCAT binds better to substrate vesicles which contain SPH; 2) SPH competes with PC in binding to the active site of the enzyme; and 3) SPH is a more powerful competitive inhibitor than a diether analog of PC. The ability of various lipoproteins to act as substrates for purified LCAT varied inversely with the SPH/PC ratio. Treatment of the lipoproteins with sphingomyelinase activated the LCAT reaction, the percent activation being directly proportional to the SPH concentration in the native lipoprotein. Enrichment of high density lipoproteins with SPH inhibited cholesterol esterification in them by 50%, and this inhibition could be reversed by the degradation of SPH. These results show that SPH is a physiological inhibitor of cholesterol esterification in the plasma, by virtue of its competition with PC, the acyl donor for the reaction.

Apolipoprotein A-I↗
PubMed1977

Regulation of plasma lecithin:cholesterol acyltransferase. II. Activation during alimentary lipemia.

The effect of dietary fat on plasma lecithin:cholesterol acyltransferase (LCAT) activity has been investigated in 14 normal male subjects. After determination of postabsorptive lipid and LCAT levels, a high-fat liquid test meal (1 to 2 gm./kg. body weight) was fed, followed by lipid and LCAT determinations at 2.5 hour intervals. Plasma triglycerides were elevated by 2.5 hours, peaked at 5.0 hours, fell at 7.5 hours, and were normalized by 10 hours. LCAT was unchanged at 2.5 hours but was elevated by 5.0 hours, exhibiting a broad plateau through 10 hours. Most subjects manifested peak responses at 7.5 hours. The mean maximal increase in individual subjects was 37.2 +/- 13.3 (S.D.) percent. LCAT changes similarly followed the elevation and recession of chylomicrons (Sf greater than 400) and very-low-density lipoprotein triglycerides, both of which closely paralleled plasma triglycerides. Enzyme responses were proportional to percentage elevations of plasma triglycerides (r = 0.93, p less than 0.01) and related to quantity of fat in the test diet. Three subjects who ingested the test diet devoid of the fat component showed no significant change in enzyme activity. Enzyme progress curves revealed linearity for 3 hours for both postabsorptive and lipemic (7.5 hour) plasma from the same subjects, supporting the validity of the assay as a measure of enzyme rate. These studies demonstrate an increase in cholesterol esterifying activity temporally related to the clearance of alimentary particles, suggesting a physiologic role in the clearance process.

Acyltransferases↗
PubMed1993

Regulation of lecithin-cholesterol acyltransferase reaction by acyl acceptors and demonstration of its "idling" reaction.

The mechanism for regulation of cholesterol esterification by lecithin-cholesterol acyltransferase (LCAT) was studied using the highly isolated enzyme from pig plasma. In the reaction with phosphatidylcholine small unilamellar vesicles, cholesterol, water, diacylglycerol, and lysophosphatidylcholine were all potent acceptors of an acyl group cleaved from the sn-2 position of egg phosphatidylcholine, generating cholesteryl ester, free fatty acid, triglyceride, and phosphatidylcholine, respectively. All of these reactions required activation by human apolipoprotein A-I, suggesting that this activation leads to the deacylation of phosphatidylcholine. Those acceptors competed against each other in this vesicle reaction system, and cholesterol was the most potent acyl acceptor. Lysophosphatidylcholine that was endogenously generated by deacylation of phosphatidylcholine in the first step of the LCAT reaction was also a good acyl acceptor, showing that the reaction is always partly "idling." Bovine serum albumin partially inhibited this idling reaction in a concentration-dependent manner up to 80% at 0.60 mM. The above results were essentially reproducible with high density lipoprotein, except that cholesterol is less potent than lysophosphatidylcholine in accepting the acyl group under the condition used. Unlike the apolipoprotein A-I-activated reaction, cholesterol was esterified only slightly by the LCAT reaction on low density lipoprotein and, consequently, did not compete against lysophosphatidylcholine for generation of phosphatidylcholine. Thus, apoB may activate LCAT in a very different manner from apoA-I. The rate of esterification of lysophosphatidylcholine on low density lipoprotein was one-tenth of that on the vesicles and on high density lipoprotein. Thus, LCAT is active on low density lipoprotein but mostly idling as deacylating and reacylating glycerophospholipids.

Acylation↗
PubMed1993

Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships.

A series of mutant apolipoprotein (apoA-I) constructs were designed and then expressed in cell culture to identify structural domains within the mature native apoA-I protein that participate in the activation of the plasma enzyme, lecithin-cholesterol acyltransferase (LCAT). Evolutionary conservation analysis has shown previously that apoA-I contains eight repeats containing 22 amino acids and two repeats containing 11 amino acids that are highly conserved among species as well as within the apolipoprotein supergene family. These tandem repeats begin at residue 44 and are usually marked by a proline residue, with six of the 22-mer repeats showing high amphipathic alpha-helical character. To determine if specific 11- or 22-amino acid domains are essential for maximal LCAT activation within the entire native protein, each of the 10 repeats was sequentially deleted using a polymerase chain reaction based method of mutagenesis. The wild-type and mutant apoA-I gene constructs were expressed in Chinese hamster ovary (CHO) cells and stable lines established. Wild-type and mutant apoA-I protein were purified from 48-96-h conditioned serum-free medium and characterized by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Wild-type apoA-I showed a single migrating band of 28,000 daltons that corresponded to the mobility of human plasma apoA-I, whereas apoA-I deletion mutants (lacking 22- or 11-mer repeats) showed the corresponding shift to lower molecular size. To measure the relative LCAT activation of all deletion mutant apoA-I proteins relative to wild-type apoA-I, an assay system utilizing small unilamellar vesicles as the lipid substrate was used. The results of these studies suggest that several central amphipathic alpha-helical regions within the mature protein are critical in LCAT activation.

Animals↗
PubMed1993

Dietary vitamin A modulates lecithin-retinol acyltransferase activity in developing chick intestine.

Retinol absorbed and generated from beta-carotene requires to be esterified by lecithin-retinol acyltransferase (LRAT) in intestinal absorptive cells. To characterize developmental changes in retinol absorptive capability in intestine, we determined LRAT activity and the amount of its retinol donor, cellular retinol-binding protein, type two (CRBP(II)) in the duodenum of developing chicks. The LRAT activity in duodenal microsomes was very low at 18- and 20-day chick embryo, but exhibited a rapid (15-fold) increase during 48 h around hatching, which occurred in parallel with the abrupt elevation of the content of CRBP(II) in chick duodenum. To examine whether dietary vitamin A affects the developmental change in LRAT activity and CRBP(II) content, 1-day-old chicks were pair-fed vitamin A-depleted or vitamin A-supplemented diet for 14 days. The chicks fed vitamin A-depleted diet showed significantly reduced LRAT activity and CRBP(II) in duodenum as early as 3 days after the start of the vitamin A-depleted diet. Changing the diet from vitamin A-depleted to vitamin A-supplemented diet led to an increase in duodenal LRAT activity within 24 h, while serum retinol concentration remained unchanged. These results suggest that duodenal LRAT activity and CRBP(II) are modulated by dietary vitamin A during the perinatal period.

Acyltransferases↗
PubMed1996

Minimally oxidized LDL is a potent inhibitor of lecithin:cholesterol acyltransferase activity.

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. As a variety of highly reactive lipid peroxidation products can transfer from oxidized LDL to HDL, we evaluated the potential deleterious effects of LDL oxidation on HDL-cholesterol metabolism. To address this issue, we exposed the HDL-containing d > 1.063 g/ml fraction of human plasma to copperoxidized LDL and assessed lecithin:cholesterol acyltransferase (LCAT) activity and apolipoproteinA-I (apoA-I) structure. To determine whether LCAT was directly affected by oxidized LDL, independent of crosslinking of apoA-I, we used an exogenous, [14C]cholesterol-labeled proteoliposome substrate to measure plasma LCAT activity. We observed an inhibition of LCAT activity where copper-oxidized LDL possessing only 2.3 +/- 0.1 and 7.3 +/- 1.4 TBARS produced 24 +/- 3% and 47 +/- 10% reductions in [14C]cholesterol esterification by 1 h, respectively. Copper-oxidized LDL that had been passed through a GF-5 desalting column, while retaining only one-third of its original TBARS, possessed nearly all of its LCAT inhibitory capacity suggesting that the LCAT inhibitory factor(s) was a lipophilic oxidation product. Analysis of polarlipids isolated from copper-oxidized LDL indicated that phospholipid and sterol fractions effectively inhibited LCAT. Copper-oxidized LDL, with as little as 6.3 TBARS, also produced intermolecular crosslinking of apoA-I molecules. Taken together, these data suggest that products of LDL oxidation may adversely affect HDL-cholesterol metabolism by two separate mechanisms: 1) a direct inhibitory effect on LCAT activity and 2) through crosslinking of apoA-I. If occurring in vivo, minimally oxidized LDL may impair cholesteryl ester formation on HDL thereby limiting the ability of HDL to function efficiently in the putative antiatherogenic reverse cholesterol transport pathway.

Apolipoprotein A-I↗