Comparison of commercial kits for apoprotein A-I and apoprotein B with standardized apoprotein A-I and B radioimmunoassays performed at the Northwest Lipid Research Center

There has recently been a proliferation of commercial kits available for apoproteins A-I and B. Since reference procedures for apoproteins have not yet been established we have elected to compare apoprotein kit methods with highly standardized apoprotein B and A-I radioimmunoassays developed at the Northwest Lipid Research Center. Commercial radial immunodiffusion kits for apoproteins A-I and B were obtained from three separate companies, Calbiochem, Daiichi Pure Chemicals, and Tago, and a commercial radioimmunoassay kit for apoprotein A-I was obtained from Ventrex Laboratories. Considerable differences were observed between the commercial kit methods and the Northwest Lipid Research radioimmunoassay methods. Some of the differences between methods were related to the assigned value of the reference materials. Other differences between methods were clearly method-dependent.     

Displacement of the human apoprotein A-I by the human apoprotein A-II from complexes of (apoprotein A-I)-phosphatidylcholine-cholesterol

Reassembly experiments, involving isolated human apoproteins A-I and A-II and (dimyristoylglycerophosphocholine)-cholesterol vesicles were performed with apoprotein mixtures at apoprotein A-I/A-II molar ratios varying between 0 and 3. The apoproteins were incubated at 24 degrees C. 28 degrees C and 32 degrees C with either pure dimyristoyl-glycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 2, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after labeling the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A-I and A-II were quantified separately by immunonephelometry. The content of apoprotein A-I was also monitored by measuring the intrinsic tryptophan fluorescence. The results suggest that apoprotein A-II has a greater affinity than apoprotein A-I for the phospholipid-cholesterol vesicles and that apoprotein A-II is able to quantitatively displace apoprotein A-I from the lipid-protein complexes. The content of apoprotein A-II in the complexes increases proportionally to the concentration of apoprotein A-II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A-II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A-I is displaced by 2 mol human apoprotein A-II.     

Comparison of two commercial nephelometric methods for apoprotein A-I and apoprotein B with standardized apoprotein A-I and B radioimmunoassays

Normotriglyceridemic and hypertriglyceridemic samples were analyzed for apoproteins A-I and B using the Beckman Array System and the Behring Nephelometer, and the nephelometric values were compared to values obtained by highly standardized radioimmunoassays developed at the Northwest Lipid Research Center. Although the means of the apoA-I values obtained by each method were similar, comparison of sample values by least-squares regression analysis revealed large differences (Sy = 20 mg/dl for Beckman, Sy = 18 mg/dl for Behring) (Sy = standard error of the estimate) regardless of whether the comparison included hypertriglyceridemic samples. For normotriglyceridemic samples, there was good agreement between apoB values obtained by the Behring Nephelometer and those obtained by RIA (r = 0.91, m = 1.03, Sy = 12 mg/dl). However, significantly higher apoB values were obtained on hypertriglyceridemic samples by the Behring Nephelometer. ApoB values for normotriglyceridemic samples obtained by the Beckman System and RIA showed fairly good correlation (r = 0.86, m = 0.71, Sy = 14 mg/dl). However, the nephelometric values for normotriglyceridemic samples averaged 29% lower than those obtained by RIA. This difference could largely be accounted for by the low apoprotein B value assigned to the Beckman calibrator. Significantly lower apoprotein B values were obtained on hypertriglyceridemic samples by the Beckman Nephelometer even after correction for calibration differences. Apoprotein values obtained by nephelometric methods may be inaccurate, particularly if the samples are hypertriglyceridemic.     

Cloning and sequencing of bovine apolipoprotein A-I cDNA and molecular evolution of apolipoproteins A-I and B-100

We have cloned and sequenced bovine apoA-I cDNA. Comparison with the apoA-I sequences of six other vertebrates shows the bovine gene to be most similar to that of the dog. Estimates of substitution rates show that apoA-I evolves approximately 25% faster than an average gene in mammalian lineages. All portions of the coding region evolve at roughly similar rates, suggesting that global conformation is conserved. However, a region of the rat protein has evolved rapidly both relative to other portions of the rat sequence and relative to homologous regions in other mammals. To extend our analysis to other apolipoproteins, we compared four vertebrate apoB-100 sequences. Conserved regions were found to include two putative LDL receptor binding domains, in addition to several regions of unidentified function. Comparison of the apoA-I sequences and the apoB-100 sequences indicates that the latter evolve approximately 40% faster than the former and at twice the average rate for mammalian proteins.     

Lipid aldehyde-mediated cross-linking of apolipoprotein B-100 inhibits secretion from HepG2 cells

Hepatic oxidative stress and lipid peroxidation are common features of several prevalent disease states, including alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), a common component of the metabolic syndrome. These conditions are characterized in part by excessive accumulation of lipids within hepatocytes, which can lead to autocatalytic degradation of cellular lipids giving rise to electrophilic end products of lipid peroxidation. The pathobiology of reactive lipid aldehydes remains poorly understood. We therefore sought to investigate the effects of 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) on the transport and secretion of very low-density lipoprotein using HepG2 cells as a model hepatocyte system. Physiologically relevant concentrations of 4-HNE and 4-ONE rapidly disrupted cellular microtubules in a concentration-dependent manner. Interestingly, 4-ONE reduced apolipoprotein B-100 (ApoB) secretion while 4-HNE did not significantly impair secretion. Both 4-HNE and 4-ONE formed adducts with ApoB protein, but 4-HNE adducts were detectable as mono-adducts, while 4-ONE adducts were present as protein–protein cross-links. These results demonstrate that reactive aldehydes generated by lipid peroxidation can differ in their biological effects, and that these differences can be mechanistically explained by the structures of the protein adducts formed.     

Tissue sites of degradation of apoprotein A-I in the rat

The tissue sites of degradation of apoprotein A-I were determined in the rat in vivo using a newly developed tracer of protein catabolism, an adduct of 125I-tyramine and cellobiose. This methodology takes advantage of the fact that when a protein labeled with 125I-tyramine-cellobiose is taken up and degraded, the radiolabeled ligand remains trapped intracellularly. Thus, radio-iodine accumulation in a tissue acts as a cumulative measure of protein degradation in that tissue. In the present studies, apoprotein AI (apo-A-I) was labeled with tyramine-cellobiose (TC). The TC-labeled apo-A-I was then reassociated with high density lipoprotein (HDL) in vivo by injection into donor animals. After 30 min, serum from donor animals was recovered and then injected into recipient rats. TC-labeled apo-A-I in the donor serum was shown to be exclusively associated with HDL. The fractional catabolic rate of 125I-TC-apo-A-I was not significantly different from that of conventionally labeled apo-A-I. The kidney was the major site of degradation, accounting for 39% of the total. The liver was responsible for 26% of apo-A-I catabolism, 96% of which occurred in hepatocytes. The kidney was also the most active organ of catabolism/g of wet weight. The tissues next most active/g of wet weight were ovary and adrenal, a finding that is compatible with a special role of HDL in the rat for delivery of cholesterol for steroidogenesis. Immunofluorescence studies of frozen sections of rat kidney demonstrated the presence of apo-A-I on the brush-border and in apical granules of proximal tubule epithelial cells. Preliminary studies using HDL labeled both with 125I-TC-apo-A-I and [3H]cholesteryl ethers again demonstrated high rates of renal uptake of apo-A-I but less than 1% of total ether uptake. It is postulated that the high activity of kidney was not due to uptake of intact HDL particles, but rather, due to glomerular filtration and tubular reabsorption of free apo-A-I.     

Effect of long-term cold exposure on apoprotein A-I, B, and E levels in the rat serum]

An increase in the quantity of triglyceride-rich VLDL as well as the total fraction VLDL and LDL occurred following the cold adaptation. The contents of apoB and apoE, respectively, increased. However, a 5-15-day exposure to cold decreased the LDL2 and apoA-1 content. In 49 days of the experiment, the LDL2 and apoA-1 content was restored.     

Comparison of deuterated leucine, valine, and lysine in the measurement of human apolipoprotein A-I and B-100 kinetics

The production rates of apolipoprotein (apo)B-100 in very low density lipoprotein and in low density lipoprotein and apolipoprotein A-I in high density lipoprotein were determined using a primed-constant infusion of [5,5,5,-2H3]leucine, [4,4,4,-2H3]valine, and [6,6-2H2,1,2-13C2]lysine. The three stable isotope-labeled amino acids were administered simultaneously to determine whether absolute production rates calculated using a stochastic model were independent of the tracer species utilized. Three normolipidemic adult males were studied in the constantly fed state over a 15-h period. The absolute production rates of very low density lipoprotein apoB-100 were 11.4 +/- 5.8 (leucine), 11.2 +/- 6.8 (valine), and 11.1 +/- 5.4 (lysine) mg per kg per day (mean +/- SDM). The absolute production rates for low density lipoprotein apoB-100 were 8.0 +/- 4.7 (leucine), 7.5 +/- 3.8 (valine), and 7.5 +/- 4.2 (lysine) mg per kg per day. The absolute production rates for high density lipoprotein apoA-I were 9.7 +/- 0.2 (leucine), 9.4 +/- 1.7 (valine, and 9.1 +/- 1.3 (lysine) mg per kg per day. There were no statistically significant differences in absolute synthetic rates of the three apolipoproteins when the plateau isotopic enrichment values of very low density lipoprotein apoB-100 were used to define the isotopic enrichment of the intracellular precursor pool. Our data indicate that deuterated leucine, valine, or lysine provided similar results when used for the determination of apoA-I and apoB-100 absolute production rates within plasma lipoproteins as part of a primed-constant infusion protocol.     

Structural comparison of human apolipoproteins B-48 and B-100

In this study we have investigated the structural relationship between human apolipoproteins B-48 and B-100 by comparing protein structure and by comparing nucleotide sequence from intestinal and hepatic cDNA clones. Sequences from intestinal and hepatic cDNA were identical over the entire distance analyzed (7194 bases), which is more than required to code for B-48. The amino-terminal amino acid sequences from intact B-48 and B-100 proteins were also identical over the entire distance analyzed (16 residues). Additional protein homology was evaluated by the combined techniques of peptide mapping and immunoblotting. Purified B-48 and B-100 were each digested with three different endoproteases, and the resulting peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide bands were then detected by silver stain and by Western blotting with antisera against specific regions of B-48 and B-100. The resulting patterns suggest that B-48 is extensively homologous with the amino-terminal portion of B-100. We have identified only four peptides from B-48 (at least one in each digest) that are absent from the parallel digests of B-100. These peptides appear to arise from the ultimate carboxyl terminus of B-48 and appear to be totally homologous with a region located near the center of B-100. Our observations suggest that mature, circulating B-48 is homologous over its entire length (estimated to be between 2130 and 2144 amino acid residues) with the amino-terminal portion of B-100 and contains no sequence from the carboxyl end of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum zinc levels and goiter in Iranian school children

Iodine deficiency has been shown to have high prevalence in Iran despite sufficient iodine supplementation. Zinc deficiency may also contribute to the pathogenesis of endemic goiter. The aim of this study was to compare serum zinc level in Iranian school children with and without goiter. A cross-sectional study was performed among urban children aged 8-12 years in city of Kerman, Iran. A multistage proportional to size cluster sampling method was used to screen 5500 subjects out of 29,787 students. After the screening phase, serum and urine specimens of randomly selected 165 students were evaluated for serum zinc levels and urinary iodine excretion and compared in goiterous and non-goiterous children. Serum zinc level was 149.5±29.4 μg/l in goiterous children and 141.2±52 μg/l in non-goiterous children but no significant difference was found between the groups (p=0.37). But urinary iodine excretion was significantly (p<0.001) lower in goiterous children (207.5 μg/l in goiterous children and 262.5 μg/l in non-goiterous children). This study showed that serum zinc level in goiterous and non-goiterus children is not different and zinc deficiency is not a risk factor for endemic goiter in this population.     

Interfacial Properties of High-Density Lipoprotein-like Lipid Droplets with Different Lipid and Apolipoprotein A-I Compositions

The surface properties of high-density lipoproteins (HDLs) are important because different enzymes bind and carry out their functions at the surface of HDL particles during metabolic processes. However, the surface properties of HDL and other lipoproteins are poorly known because they cannot be directly measured for nanoscale particles with contemporary experimental methods. In this work, we carried out coarse-grained molecular dynamics simulations to study the concentration of core lipids in the surface monolayer and the interfacial tension of droplets resembling HDL particles. We simulated lipid droplets composed of different amounts of phospholipids, cholesterol esters (CEs), triglycerides (TGs), and apolipoprotein A-Is. Our results reveal that the amount of TGs in the vicinity of water molecules in the phospholipid monolayer is 25–50% higher compared to the amount of CEs in a lipid droplet with a mixed core of an equal amount of TG and CE. In addition, the correlation time for the exchange of molecules between the core and the monolayer is significantly longer for TGs compared to CEs. This suggests that the chemical potential of TG is lower in the vicinity of aqueous phase but the free-energy barrier for the translocation between the monolayer and the core is higher compared to CEs. From the point of view of enzymatic modification, this indicates that TG molecules are more accessible from the aqueous phase. Further, our results point out that CE molecules decrease the interfacial tension of HDL-like lipid droplets whereas TG keeps it constant while the amount of phospholipids varies.     

Presence of B-100 in rat mesenteric chyle

Molecular forms of apolipoprotein B (ApoB) were studied in the rat intestinal chyle by SDS-polyacrylamide gel electrophoresis, immunoblotting and immunodiffusion. Time studies on intestinal chyle showed the presence of B-100 in all the samples analyzed within 3 hr after drawing. However, the analyses repeated on day 2 or day 3 revealed disappearance of B-100 and appearance of B-48. Addition of 3 mM EDTA, 10 mM diisopropylfluorophosphate, 5 mM chloroquine and 10 mM epsilon-amino caproic acid slowed down but could not prevent the disappearance of B-100. Chylomicrons isolated from chyle in the presence of preservatives immediately after drawing displayed B-100 as a major and B-48 as a minor ApoB form. However, repeatedly washed chylomicrons or those isolated from chyle 18-24 hr after drawing showed B-48 as the only ApoB present. These results suggest that rat intestine synthesizes B-100 which is quickly converted to smaller molecular form.     

Stimulation of arterial smooth muscle cell proliferation by remnant lipoprotein particles isolated by immuno-affinity chromatography with anti-apo A-I and anti-apo B-100.

Postprandial lipidemia, characterized by high plasma triglyceride-rich lipoprotein remnants, is associated with atherosclerosis. It has also been known that proliferation of vascular smooth muscle cells is crucial for the development of atherosclerosis. In this study, we investigated the direct effect of remnant lipoprotein particles, which consist of chylomicron remnants and very low density lipoprotein remnants, on vascular smooth muscle cell proliferation. Blood was collected from six patients with postprandial lipidemia two hours after their usual meal. Remnant lipoprotein particles were isolated from plasma by immuno-affinity chromatography containing two monoclonal antibodies, anti-apo A-I (H-12) and anti-apo B-100 (JI-H). Remnant lipoprotein particles, as well as betaVLDL, significantly stimulated the proliferation of porcine coronary artery smooth muscle cells in a concentration-dependent manner, whereas very low density lipoprotein (d < 1.006) was virtually ineffective. These observations are consistent with recent reports that triglyceride-rich lipoprotein remnants, which are rich in apo E as well, are atherogenic.     

Identification of sequence homology between human plasma apolipoprotein B-100 and apolipoprotein B-48

The structural relationship between apolipoprotein B-100 (apo-B-100) and apolipoprotein B-48 (apo-B-48) has not been elucidated. A peptide fragment (MDB-18) of approximately 6 kDa was isolated from a tryptic digest of apo-B-100. The sequence of the first 22 amino acids of MDB-18 was determined by Edman degradation. A 15-residue peptide corresponding to this sequence was synthesized by the solid-phase method and was utilized to develop a sequence-specific polyclonal antibody. On immunoblot analysis, the antibody recognized both intact apo-B-100 and apo-B-48. In addition, preincubating the antibody with the synthetic peptide abolished the recognition of both apo-B-100 and apo-B-48. These data are interpreted as indicating that there is an amino acid sequence homology between apo-B-100 and apo-B-48. Since the MDB-18 peptide is located in the carboxyl region of the B-100 molecule, we propose apo-B-100 and apo-B-48 share a common carboxyl region sequence.     

Familial defective apolipoprotein B-100: a lesson from homozygous and heterozygous patients

Familial defective apolipoprotein B-100 (FDB) is a genetic disorder caused by a substitution of glutamine for arginine at residue 3500 of the apolipoprotein B-100 molecule. We have identified 23 heterozygotes and one homozygote for FDB (frequency 1:20) in a group of 510 patients with hypercholesterolemia. Mean age of the patients (18 females and 6 males) was 46 years. The diagnosis of FDB was based on point mutation PCR analysis of exon 26 of the apo B gene. Plasma lipids in heterozygous patients were: total cholesterol 8.76+/-1.2 mmol/l, triglycerides 1.42+/-0.5 mmol/l, HDL-cholesterol 1.43+/-0.3 mmol/l, LDL-cholesterol 6.69+/-1.2 mmol/l, apoB 1.69+/-0.4 g/l, Lp(a) 0.26+/-0.2 g/l. The most frequent apoE genotype was 3/3 (19 patients), apoE 3/4 genotype was found in 3 patients and one person had apoE 2/3. Xanthelasma palpebrarum was present in 4 patients and tendon xanthomas in 3 patients including the homozygote. Premature manifestation of coronary heart disease was revealed in 3 patients. Sixteen patients were treated with statins, a combination of statin and resin was used in 2 patients (including the homozygote), whereas six patients were treated with the diet only. We conclude that although the plasma lipid levels of total and LDL cholesterol in FDB patients are lower than in patients with familial hypercholesterolemia, the patients with FDB suffer from premature atherosclerosis. The therapeutic approach to FDB individuals and patients with familial hypercholesterolemia is very similar.     

Structure of apolipoprotein B-100 in low density lipoproteins

There is general consensus that amphipathic alpha-helices and beta sheets represent the major lipid-associating motifs of apolipoprotein (apo)B-100. In this review, we examine the existing experimental and computational evidence for the pentapartite domain structure of apoB. In the pentapartite nomenclature presented in this review (NH(2)-betaalpha(1)-beta(1)-alpha(2)-beta(2)-alpha(3)-COOH), the original alpha(1) globular domain (Segrest, J. P. et al. 1994. Arterioscler. Thromb. 14: 1674;-1685) is expanded to include residues 1;-1,000 and renamed the betaalpha(1) domain. This change reflects the likelihood that the betaalpha(1) domain, like lamprey lipovitellin, is a globular composite of alpha-helical and beta-sheet secondary structures that participates in lipid accumulation in the co-translationally assembled prenascent triglyceride-rich lipoprotein particles. Evidence is presented that the hydrophobic faces of the amphipathic beta sheets of the beta(1) and beta(2) domains of apoB-100 are in direct contact with the neutral lipid core of apoB-containing lipoproteins and play a role in core lipid organization. Evidence is also presented that these beta sheets largely determine LDL particle diameter. Analysis of published data shows that with a reduction in particle size, there is an increase in the number of amphipathic helices of the alpha(2) and alpha(3) domains associated with the surface lipids of the LDL particle; these increases modulate the surface pressure decreases caused by a reduction in radius of curvature. The properties of the LDL receptor-binding region within the overall domain structure of apoB-100 are also discussed. Finally, recent three-dimensional models of LDL obtained by cryoelectron microscopy and X-ray crystallography are discussed. These models show three common features: a semidiscoidal shape, a surface knob with the dimensions of the betaC globular domain of lipovitellin, and planar multilayers in the lipid core that are approximately 35 A apart; the multilayers are thought to represent cholesteryl ester in the smectic phase. These models present a conundrum: are LDL particles circulating at 37 degrees C spheroidal in shape, as generally assumed, or are they semidiscoidal in shape, as suggested by the models? The limited evidence available supports a spheroidal shape.     

Plasma selenium levels in healthy blood bank donors in the central-eastern part of Belgium

Graphite furnace atomic absorption spectrometry, with Zeeman background correction and after improved matrix modification, was used to measure the plasma selenium content of healthy blood bank donors in the central part of Belgium.

The mean plasma selenium concentration of 80 men and 80 women was 79.7±4.4 ng/mL with a range of 55.0–117.4 ng/mL.

There was no gender difference observed. Plasma selenium level was significantly highest for the adult group, aged 45–64 years, compared to the others, except the young adults (18–24 years).

The mean plasma selenium concentration measured corresponded well with literature data for Belgium. The obtained values were found to be in the medium range, compared with recent literature values for the European countries.