The effect of dietary copper on rat plasma apolipoprotein B,E plasma levels,and apolipoprotein gene expression in liver and intestine

The plasma levels of apo B and apo E, and the level of hepatic and intestinal mRNA coding for these apolipoproteins were investigated in weanling male rats pair-fed for 6 wk with a control or copperdeficient diet. Plasma cholesterol, triglycerides, and phospholipids were significantly increased, and plasma apo B and apo E levels were also markedly increased in copper-deficient rats as compared to control rats. Copper deficiency significantly increased triglyceride levels and decreased cholesterol levels in the liver. No major differences in the levels of hepatic and intestinal apo B and apo E mRNA occurred between control and copper-deficient rats. These data imply that hypertriglyceridemia dn hypercholesterolemia owing to the copper deficiency are not accompanied by modifications in the gene expression at the mRNA level in the liver and intestine of the apolipoproteins studied.     

Apolipoprotein E polymorphism in the Netherlands and its effect on plasma lipid and apolipoprotein levels

Summary By isoelectric focusing of delipidated sera followed by immunoblotting we studied the apolipoprotein (apo) E polymorphism in 2018 randomly selected 35-years-old males from three different areas in the Netherlands. Comparison of the APOE allele (E^*2, E^*3, and E^*4) frequencies estimated in this study with those reported for several other population samples showed that there are marked differences between the Dutch population and the populations of Japan, New Zealand, Finland, and the United States. These differences in APOE allele frequencies appeared to be mainly due to differences in frequencies of the E^*2 allele (decreased in Japan and Finland; increased in New Zealand) and the E^*4 allele (increased in Finland; decreased in Japan and the United States). No difference in APOE allele frequencies was found between the Dutch population and the populations of West Germany and Scotland. Measurements of plasma cholesterol and apo B and E concentrations showed that the E^*4 allele is associated with elevated plasma cholesterol and apo B levels and with decreased apo E concentrations, whereas the opposite is true for the E^*2 allele. In the Dutch population, the sum of average allelic effects of the common APOE alleles on plasma cholesterol and apo B levels is 6.8% and 14.2%, respectively, of the total population mean. The total average allelic effect on plasma apo E concentrations was more pronounced (50.1%), suggesting that the APOE alleles primarily affect apo E concentrations rather than plasma cholesterol and apo B levels. This hypothesis is sustained by the observation that for plasma apo E levels the genetic variance associated with the APOE gene locus contributed about 18% to the total phenotypic variance. For plasma cholesterol and apo B this contribution was only 1.4% and 2.3% and is relatively low as compared with that reported for other population samples.     

Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epitopes of the inhibiting antibodies, and finally to examine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, designated 1D7, 6C5, and 3H1, were found to inhibit binding, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measurement of the heparin binding activity of fragments containing epitopes of the inhibiting antibodies demonstrated that apo-E3 contains two heparin binding sites. The first site is located in the vicinity of residues 142-147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal region of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.     

Determination of the rheumatoid factor in gouty patients

The authors have examined the incidence of the rheumatoid factor in the serum of a group of 100 patients, 95 males and 5 females, suffering from gout, of whom only 6 suffered from chronic gout. The rheumatoid factor in the serum was measured by RA-test (1:40) and by Waaler-Rose test (1:32). The rheumatoid factor was not present in the examined subjects and so we can state that such serum parameter does not represent, for its negativity, a laboratory index of any evidence in the gout and it doesn't represent a reason for diagnostic doubts with other forms with the rheumatoid factor in the serum.     

Characterization of apolipoprotein E genetic variations in Taiwanese: association with coronary heart disease and plasma lipid levels

Apolipoprotein E (apoE, protein; APOE, gene) is important in lipoprotein metabolism. Three isoforms, apoE2 (Cys112 Cys158), apoE3 (Cys112 Arg158), and apoE4 (Arg112 Arg158), are present in the general population. This report investigates the frequency distribution of apoE isoforms and the association of APOE genotypes with plasma lipid profile and coronary heart disease (CHD) in a population of Taiwan. ApoE isoforms were determined genetically by polymerase chain reaction and HhaI restriction enzyme digestion in control and coronary heart disease (CHD) patients. Plasma lipid and lipoprotein concentrations were also determined. The control group exhibited frequencies of 84.6% APOE3, 7.9% APOE4, 7.5% APOE2, 70.6% APOE3E3, 14.4% APOE3E4, 13.6% APOE2E3, and 1.4% APOE2E4. Comparable frequencies were observed in the CHD group. In both APOE2 carrier and APOE3E3 groups, the CHD patients expressed abnormal lipid profiles while the control group expressed normal lipid profiles. The APOE4 carriers, however, expressed abnormal lipid profiles in both normal control and CHD groups. Extremely high apoE levels in the hypertriglyceridemic group (TG > 400 mg/dL) seemed to be undesirable and were often observed in CHD patients.     

Determination of human apolipoprotein E by electroimmunoassay.

1. Apolipoprotein E ("arginine-rich" polypeptide) was isolated from delipidized human very low density lipoproteins by agarose column chromatography in the presence of 6 M guanidine-hydrochloride. 2. An electroimmunoassay ("rocket" electrophoresis) is described for quantitative determination of human serum apolipoprotein E. Purified apolipoprotein E was used for the preparation of monospecific antisera and standardization of assay. This sensitive, specific, rapid (time required for the completion of the assay is 5 h) and precise (the within- and between-assay coefficients of variation are 5 and 8%, respectively) assay is applicable to measurement of apolipoprotein E in whole serum and density classes. The results correlated well with those obtained by radial immunodiffusion (r = 0.85). 3. Serum apolipoprotein E levels of normal subjects and hyperlipoproteinemic phenotypes IIa, IIb and IV were the same (10 to 16 mg/100 ml). In contrast, patients with type III and V hyperlipoproteinemias had markedly elevated serum apolipoprotein E levels )27 and 25 mg/100 ml, respectively). The apolipoprotein E in serum of normolipidemic subjects was equally distributed among three major lipoprotein density classes: d less than 1.030 g/ml (27%), d 1.030-1.063 g/ml (36%)and d 1.063-1.21 g/ml (37%).     

Sex-related differences in the concentrations of apolipoprotein E in human blood plasma and plasma lipoproteins

Apolipoprotein E (apoE) and lipoprotein cholesterol and triglycerides were measured in blood serum of 272 persons randomly selected from a large industrial population in northern California. Serum apoE level increased linearly by 0.013 mg/dl with each 1 mg/dl increase in very low density lipoprotein (VLDL) triglycerides. This estimate was independent of sex and the use of sex hormones by women. Compositional studies of isolated apoVLDL in 156 hypertriglyceridemic men and 162 normotriglyceridemic persons of both sexes from the same population also indicated that the content of apoE was independent of VLDL level, sex, and hormone use. The estimate of the relationship between serum apoE and VLDL-triglycerides derived from these compositional studies was comparable to that derived by regression analysis. Regression analysis also indicated that only 10-20% of the apoE in the serum of the average person is in the VLDL fraction. Serum apoE levels were 1.4 mg/dl higher in women than in men with the same VLDL-triglyceride level and 1.8 mg/dl lower in women using contraceptive drugs than in nonusers of like age and VLDL-triglyceride level.     

Serum levels of apolipoprotein A-I, A-II and HDL-cholesterol in second half of normal pregnancy and in pregnancy complicated by pre-eclampsia

Serum concentrations of Apolipoprotein A-I and A-II, (Apo A-1 and Apo A-II) HDL-cholesterol (HDL-C), Total Cholesterol (TC), triglycerides (TG) and lipoprotein electrophoresis were assayed serially in the second half of normal pregnancy (21 women), in pre-eclampsia (26 women) and in both groups one and six weeks after delivery. In the normal group we found increased concentrations of Apo A-I and HDL-C, which remained unaltered during pregnancy. Apo A-II was unchanged. Correlation coefficients for Apo A-II vs HDL-C and Apo A-I vs Apo A-II decreased gradually towards delivery while it remained at an elevated and unaltered level for Apo A-I vs HDL-C. The Apo A-I/HDL-C ratio was unaltered during the whole study while the Apo A-I/A-II ratio was elevated during pregnancy and the Apo A-II/HDL-C ratio was reduced. These results may indicate a gradual change in the surface structure of the HDL particle or its subfractions. In pre-eclampsia Apo A-I and HDL-C concentrations were reduced, TG was increased and Apo A-II and TC were unchanged when compared with the normal pregnancy group. A more pronounced correlation coefficient was recorded for Apo A-I vs HDL-C than for Apo A-II vs HDL-C and Apo A-I vs Apo II. The results indicate that from an atherogenic point of view normal pregnancy seems more beneficial than pre-eclampsia.     

Accumulation of human apolipoprotein E in the plasma of transgenic mice

Three separate lines of transgenic mice were created with integrated copies of an 11.1-kilobase pair human DNA fragment containing the apolipoprotein (apo) E gene. The endogenous mouse apoE gene is primarily expressed in the liver with varying levels of expression in other tissues. However, in all three transgenic lines high levels of human apoE mRNA were detected only in the kidney, with lower levels found in the liver and other tissues; despite this profile of human apoE mRNA, human apoE was found in the plasma of the transgenic mice at levels comparable to those found in human plasma. All of the human apoE in the plasma of the transgenic mice was associated with lipoproteins. These results suggest that the domain responsible for the high level of apoE expression in liver lies outside of the microinjected DNA fragment and that an ectopic site of expression of an introduced gene may be permissive for the accumulation of its protein in plasma.     

Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice

The apolipoprotein E gene knockout (apoE-/-) mouse develops atherosclerosis that shares many features of human atherosclerosis. Increased levels of glycosphingolipid (GSL) have been reported in human atherosclerotic lesions; however, GSL levels have not been studied in the apoE-/- mouse. Here we used HPLC methods to analyze serum and aortic GSL levels in apoE-/- and C57BL/6J control mice. The concentrations of glucosyl ceramide (GlcCer), lactosyl ceramide (LacCer), GalNAcbeta1-4Galbeta1-4Glc-Cer (GA2), and ceramide trihexoside (CTH) were increased by approximately 7-fold in the apoE-/- mouse serum compared with controls. The major serum ganglioside, N-glycolyl GalNAcbeta1-4[NeuNAcalpha2-3]Galbeta1-4Glc-Cer (N-glycolyl GM2), was increased in concentration by approximately 3-fold. A redistribution of GSLs from HDL to VLDL populations was also observed in the apoE-/- mice. These changes were accompanied by an increase in the levels of GSLs in the aortic sinus and arch of the apoE-/- mice. The spectrum of gangliosides present in the aortic tissues was more complex than that found in the lipoproteins, with the latter represented almost entirely by N-glycolyl GM2 and the former comprised of NeuNAcalpha2-3Galbeta1-4Glc-Cer (GM3), GM2, N-glycolyl GM2, GM1, GD3, and GD1a. In conclusion, neutral GSL and ganglioside levels were increased in the serum and aortae of apoE-/- mice compared with controls, and this was associated with a preferential redistribution of GSL to the proatherogenic lipoprotein populations. The apoE-/- mouse therefore represents a useful model to study the potential role of GSL metabolism in atherogenesis.     

Isolation and partial characterization of an arginine-rich apolipoprotein from human plasma very-low-density lipoproteins: apolipoprotein E.

A water-insoluble apoprotein was isolated from apo-VLDL by column chromatography on Sephadex G-200 in sodium dodecylsulfate followed by preparative polyacrylamide gel electrophoresis in a discontinous sodium dodecylsulfate system, or by preparative electrophoresis alone. The protein was similar in amino acid composition to the "arginine-rich protein" reported by Shore and Shore. It represented about 10% of the total protein mass of VLDL. The apoprotein showed one single band with an apparent Mr of 39000 in sodium dodecylsulfate gel electrophoresis, and was homogeneous in gel electrophoresis at pH 8.9 In 8M urea. Immunochemical studies also showed homogeneity of this protein, and antisera prepared against it did not react with any other of the well known apolipoproteins, but did react with VLDL and apo-VLDL preparations. Analytical isoelectric focusing in 8M urea resulted in a heterogeneous banding pattern showing three major polypeptides with pI values of 5.5, 5.6 and 5.75. Thus this apolipoprotein clearly differs from the apo-B and apo-C polypeptides of VLDL as well as from apoproteins A and D in its molecular weight, amino acid composition, focusing behavior and immunochemical properties.     

Localization of apolipoprotein E receptor 2 to caveolae in the plasma membrane

The LDL receptor (LDL-R) promotes the specific endocytosis and lysosomal delivery of extracellular lipoprotein ligands via clathrin-coated pits. It was widely assumed that other closely related members of the LDL-R gene family would have similar functions, but recent experimental evidence has revealed that one such protein, apolipoprotein E receptor 2 (apoER2), has a critical role as an "outside-in" signal transducer in the brain. ApoER2 signaling appears to require interaction between its cytoplasmic domain and adapter molecules such as Dab1, JIP 1 and JIP 2, and PSD-95. Many of the receptors for other signaling pathways affected by such adapter molecules are compartmentalized into specialized microdomains within the plasma membrane termed caveolae. Here, we show that apoER2, but not LDL-R, is localized to caveolae, supporting the concept that its physiological role is in cell signaling, rather than in endocytosing ligands.     

Structural and functional basis for increased HDL-cholesterol levels due to the naturally occurring V19L mutation in human apolipoprotein A-I

Several hereditary point mutations in human apolipoprotein A-I (apoA-I) have been associated with low HDL-cholesterol levels and/or increased coronary artery disease (CAD) risk. However, one apoA-I mutation, the V19L, recently identified in Icelanders, has been associated with increased HDL-cholesterol levels and decreased CAD risk. In an effort to gain mechanistic insight linking the presence of this mutation in apoA-I with the increase of HDL-cholesterol levels we evaluated the effect of V19L mutation on the conformational integrity and functional properties of apoA-I in lipid-free and lipidated form. ApoA-I[V19L] was found to be thermodynamically destabilized in lipid-free form and displays an increased capacity to associate with phospholipids compared to WT apoA-I. When associated to reconstituted HDL (rHDL), apoA-I[V19L] was more thermodynamically stabilized than WT apoA-I. ApoA-I[V19L] displayed normal capacity to promote ABCA1-mediated cholesterol efflux and to activate the enzyme LCAT, in lipid-free and rHDL-associated forms, respectively. Additionally, rHDL-associated apoA-I[V19L] showed normal capacity to promote ABCG1-mediated cholesterol efflux, but 45% increased capacity to promote SR-BI-mediated cholesterol efflux, while the SR-BI-mediated HDL-lipid uptake was normal. Overall, our findings show that the apoA-I V19L mutation does not affect the first steps of HDL biogenesis pathway. However, the increased capacity of apoA-I[V19L] to associate with phospholipids, in combination with the enhanced thermodynamic stability of lipoprotein-associated apoA-I[V19L] and increased capacity of apoA-I[V19L]-containing lipoprotein particles to accept additional cholesterol by SR-BI could account for the increased HDL-cholesterol levels observed in human carriers of the mutation.     

Dietary fat and apolipoprotein genotypes modulate plasma lipoprotein levels in Brazilian elderly women

Studies show that genetic polymorphisms in apolipoproteins, which are in charge of lipid transport, predispose to atherogenic dyslipidemia. This study aimed to investigate the impact of apolipoprotein E, A5, and B genotypes and dietary intake on lipid profile in a sample of elderly women in Brazil. Two hundred and fifty-two women (60 years or older) living in the outskirts of the Brazilian Federal District underwent clinical and laboratory assessments to characterize glycemic and lipidemic variables, and also to exclude confounding factors (smoking, drinking, hormone replacement, cognitive impairment, physical activity). Three-day food records were used to determine usual dietary intake, whereas genotypic evaluations were in accordance to established methodologies. Genotype frequencies were consistent with the Hardy–Weinberg equilibrium. Prior to adjustment, individuals carrying the ε2 allele showed higher serum levels of triglycerides (P < 0.05) and VLDL (P < 0.005) compared to ε4 carriers, whereas LDL levels were considerably elevated in ε4 compared to ε2 carriers. In the presence of high intake of total fat or a low ratio of polyunsaturated to saturated fatty acid, ε4 carriers lost protection against hypertriglyceridemia. There was no association of the apolipoprotein A5 and B genotypes with lipidemic levels independently of the fat intake regimen. Results are suggestive of a dysbetalipoproteinemic-like phenotype in postmenopausal women, with remarkable gene–diet interaction.     

Phenotyping of human apolipoprotein E from whole blood plasma by immunoblotting

Human apolipoprotein E (apoE) exists in the population in three common genetically determined isoforms, apoE-2, E-3, and E-4, that are coded for by three alleles epsilon-2, epsilon-3 and epsilon-4 at the apoE structural gene locus resulting in six phenotypes, three homozygotes (E 2/2, E 3/3, and E 4/4) and three heterozygotes (E 2/3, E 2/4, and E 3/4). A new procedure is described that allows identification of apoE isoforms and phenotypes from whole plasma or serum without the need for isolating apoE-containing lipoproteins or two-dimensional gel electrophoresis of serum. This rapid method combines cysteamine treatment of apoE in plasma, separation in parallel of cysteamine-treated and untreated hydrophobic serum proteins by charge-shift electrophoresis, and isoelectric focusing of apolipoproteins with immunoblotting. Compared to phenotyping of apoE after isolation of VLDL, the new procedure agreed in most cases and may be of special value in detecting apoE mutants that differ in their cysteine residues or either are spun off during isolation of lipoproteins or cofocus with other apoproteins and thus escape detection by conventional one-dimensional techniques. The method provides a simple tool to screen apoE isoforms that are known to have a major impact on individual plasma cholesterol levels.     

The effect of apolipoprotein E polymorphism on plasma cholesteryl ester transfer protein activity in type 2 diabetic patients

We studied the relationship between apo E polymorphism and cholesteryl ester transfer protein (CETP) activity in 127 type 2 diabetic patients who did not take lipid lowering drugs. Furthermore, we studied the relationship between apo E and cholesteryl ester transfer protein (CETP) in modulating plasma triglyceride and HDLcholesterol. Apo E genotypes were determined by PCR-RFLP, and CETP activity was measured using an exogenous way. Our results showed that the CETP activity increased significantly in the E2 carrier group compared to E4 carriers and E3/E3 homozygous (84.7 ± 43.9 vs. 62.5 ± 35.9 vs. 52.6 ± 23.6 nmol CE/ml/2h, respectively; p = 0.015). However, there was no association between apo E polymorphism and lipid parameter variations. Even after adjustment for CETP activity, the results remained unchanged, showing that CETP did not step in the relationship between apo E and lipid parameter variations. In conclusion there is an association between apo E polymorphism and CETP activity, and this association did not affect the relationship between apo E polymorphism and triglyceride and HDLcholesterol concentrations. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 6, pp. 931–935. The text was submitted by the authors in English.