Biomedical subjects

M Rosseneu

Publications and source records attributed to M Rosseneu.

At least 109 records · Page 6Linked to original sources

PubMed1992

Acquired hypolipoproteinemia.

We present a six-year follow-up of a boy with a novel type of hypolipoproteinemia, with clinical and biochemical features distinct from classical hypoalphalipoproteinemias. There were abnormally low concentrations of total and high-density lipoprotein (HDL) cholesterol, apolipoprotein (apo) B, apo A-I, and apo A-II, and the phospholipids were decreased. The most striking abnormality was an extra fraction containing mainly phospholipids and apo A-I in the HDL3 subfraction. This fraction is reminiscent of concentric 20- to 50-nm-diameter lamellar phospholipid liposomes. Plasma lecithin:cholesterol acyltransferase activity was strongly decreased. We noted a persisting polyclonal hypergammaglobulinemia, hematological abnormalities (hemolytic anemia and thrombocytopenia), and a progressive splenomegaly. After the five-year follow-up, the patient had recurrent severe infections; moderate hematuria and proteinuria developed gradually. Treatment with corticosteroids and immunoglobulins improved thrombocytopenia and hypolipoproteinemia. These clinical and biochemical findings differ from those in the known primary and secondary hypo-alpha-lipoproteinemia syndromes. Although investigation of the relatives suggests a familial predisposition for hypo-alpha-lipoproteinemia, the subject's condition can be regarded as acquired.

Adolescent↗

PubMed1991

Catalytic triad residue mutation (Asp156----Gly) causing familial lipoprotein lipase deficiency. Co-inheritance with a nonsense mutation (Ser447----Ter) in a Turkish family.

We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity. We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors. Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues. Both patients were homozygous for both mutations. Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations. In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector. Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant. Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly. Immunoreactive LPLs were produced in all cases. The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients. The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity. Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity. We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance. Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗

PubMed1991

Lipoprotein(a) profiles and evolution in newborns.

Plasma Lp(a) concentrations in newborns were quantified by a specific and sensitive ELISA assay and their evolution was followed between birth and 6 months. The influence of the diet on Lp(a) levels was also investigated. Moreover, the high sensitivity of the assay enabled the localisation of the Lp(a) fraction in the lipoprotein profile obtained after plasma separation by gel chromatography. Lp(a) levels are low at birth and rise significantly between 0 and 7 days post partum; in this newborn population, a continuous rise of the mean Lp(a) levels was observed until 180 days, in contrast with the apo B concentration that plateaus after 7 days. An early screening enabled the detection of newborns with elevated Lp(a) levels compared to the mean value of their age group. A further follow-up of some cases at 16 months confirmed the high Lp(a) levels measured in the infants and at least one of the parents. The investigation of the lipoprotein profiles as a function of the age of the newborn enabled an estimation of the size and distribution of the Lp(a) lipoprotein in four infants. At birth, Lp(a) particles were larger than LDL and tend to become more heterogeneous with increasing age of the newborn. We could not observe any statistically significant influence of the nutritional factors on the plasma Lp(a) concentrations at any age.

Age Factors↗

PubMed1991

Effect of simvastatin treatment on the dyslipoproteinemia in CAPD patients.

HMG-CoA reductase inhibitors have been proven effective in decreasing the plasma cholesterol levels in patients affected with various forms of hypercholesterolemia, familial dysbetalipoproteinemia, familial combined hyperlipidemia and in nephrotic and diabetic dyslipidemia. The purpose of this study was to monitor and evaluate the efficiency and safety of the therapy with simvastatin, an HMG-CoA reductase inhibitor, in a group of patients treated by continuous ambulatory peritoneal dialysis (CAPD) with severe hypercholesterolemia. Monitoring of the changes occurring in the various lipids and apolipoproteins in these patients included the measurements of the plasma lipids and apolipoproteins A-I, A-II, B, C-II, A-IV and Lp(a). Lipoproteins were separated by gel filtration, on a Superose 6HR column, before and after 24 weeks of treatment. The patterns were compared to those observed in a group of primary hyperlipidemic patients treated with Lovastatin, a compound of the same class. The drug was well tolerated by the CAPD patients and no adverse reaction was observed. In addition to the decrease of the total and LDL cholesterol, similar to that reported in other groups of patients, we further observed a decrease of the apo E concentration in both the CAPD and the hyperlipidemic patients. This decrease was especially pronounced in the HDLE fraction and could involve an upregulation of the apo B-E and/or apo E receptor. These results should provide information about the mechanism of action of this drug in patients with end-stage renal disease.

Adult↗

PubMed1991

Characterization of the discoidal complexes formed between apoA-I-CNBr fragments and phosphatidylcholine.

The structure, composition, and physico-chemical properties of lipid-protein complexes generated between dimyristoylphosphatidylcholine (DPMC) and the CNBr fragments of human apoA-I were studied. The fragments were separated by high performance liquid chromatography and purified on a reversed-phase column. The complexes with DMPC were isolated on a Superose column; their dimensions were obtained by gradient gel electrophoresis and by electron microscopy. The secondary structure of the protein in the complexes was studied both by circular dichroism and by attenuated total reflection infrared spectroscopy. The fragments 1 and 4 of apoA-I, containing, respectively, two and three amphipathic helices, recombined with the phospholipid to generate discoidal particles with sizes similar to that of apoA-I- and apoA-II-DMPC complexes. The infrared measurements indicated that in all complexes the apolipoprotein helical segments were oriented parallel to the phospholipid acyl chains and that the protein was located around the edges of the discs. Computer modelling of the complexes based on energy minimization techniques proposed a model for these particles in agreement with the dimensions measured experimentally. In conclusion, we propose that apoA-I and its longest CNBr fragments are able to generate discoidal particles with DMPC, with apolipoprotein helical segments oriented parallel to the acyl chains of the phospholipids.

Apolipoprotein A-I↗

PubMed1991

Quantification of lipoprotein(a) in dried blood spots and screening for above-normal lipoprotein(a) concentrations in newborns.

Lipoprotein(a) [Lp(a)] is considered an additional, independent, and largely genetically determined risk factor for the development of premature coronary heart disease. Analogous with increased Lp(a) concentrations that represent an additional risk factor in adults, above-normal concentrations of Lp(a) can be detected in five- to seven-day-old newborns. We describe a simple enzyme-linked immunosorbent assay for measuring Lp(a) in dried blood spots collected by heel-prick in five- to seven-day-old infants. Lp(a) could be quantitatively recovered from blood spots. We chose a cutoff value of 100 mg/L for identifying the newborns at risk, based on the Lp(a) distribution in 180 such infants.

Apolipoproteins A↗

PubMed1990

Mode of assembly of amphipathic helical segments in model high-density lipoproteins.

The structure of discoidal apo A-I-phospholipid complexes, representing the metabolic precursors of mature high-density lipoprotein particles, was studied by a combination of both a theoretical and an experimental approach. The secondary structure of the complex was determined by circular dichroic measurements, while the relative orientation of the apo A-I helical segments and of the phospholipid acyl chains was determined by ATR infrared measurements. Fluorescence energy transfer between the tryptophan residues of apo A-I and fluorescent phospholipid probes yielded an estimation of the relative topography of the lipid and apolipoprotein components in discoidal and spherical particles. The theoretical approach consisted of the identification of the helical segments in various apo A-I species. These segments were then oriented at a lipid/water interface by minimization of their hydrophobic and hydrophilic transfer energies. The calculation of the hydrophobicity profiles along the axis of the helices leads to the identification of specific interactions between pairs of helices. The helices were further assembled together with the phospholipids by computer modelling, enabling an estimation of the dimensions of the complex. The combination of the experimental and theoretical results yielded a model for discoidal apolipoprotein-phospholipid complexes, in which the amphipathic helical segments are oriented along the edges of the discs. Such a model can be extended to the conversion of these complexes into mature spherical HDL, through the formation of a cholesteryl ester core.

Amino Acid Sequence↗

PubMed1990

Determinants of fat consumption in a general population.

In a random sample of the adult population (aged 25-64 years) from Ghent and Charleroi in Belgium, dietary fat consumption was estimated using both qualitative and quantitative dietary methods. A fat consumption score (FCS) was constructed from the qualitative method, and it correlated significantly with the dietary polyunsaturated/saturated fatty acids (P/S) ratio calculated from the quantitative method (r = -0.48; p less than 0.001). Differences in FCS were then studied using univariate and multivariate analyses. In both men and women, FCS was higher in Charleroi than in Ghent. In men, FCS was inversely related with educational level and with age. In women, significant correlations were observed with marital status. No differences were found in FCS by smoking behaviour. With the coronary risk factors, low-order relationships were found, but only in men. In the middle-aged group, significant correlations were found between FCS and the fatty acid composition of the serum cholesteryl esters, in particular with the linoleate/oleate ratio.

Adult↗

PubMed1990

Immunological assays of apolipoproteins in plasma: methods and instrumentation.

A number of immunological techniques--radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), electroimmunoassay, radial immunodiffusion, and a variety of immunoprecipitin assays--have been used to quantify apolipoproteins in plasma. This paper outlines their technical details and discusses their major advantages and drawbacks. The most sensitive procedures, RIAs and ELISAS, are best suited to quantifying those apoproteins found in low concentration in plasma. Immunoturbidimetric assays, on the other hand, which are readily automated, are being widely used to quantify apolipoproteins A-I and B. Apolipoprotein quantification is complicated by the interaction of the proteins with lipids, which can often mask their antigenic determinants. This problem may be circumvented by pretreatment of the samples, by selection of appropriate standards, or by the use of polyclonal or monoclonal antibodies that interact with permanently exposed epitopes on the lipoproteins' surfaces. Our proposed methods for measurement of the individual apolipoproteins give consideration to these approaches.

Apolipoproteins↗

PubMed1989

Evaluation of the secondary structure of apo B-100 in low-density lipoprotein (LDL) by infrared spectroscopy.

The secondary structure of the apo B-100 protein present in human low density lipoprotein has been investigated by transmission and attenuated total reflection infrared spectroscopy. The amount of beta-sheet (41%) is significantly higher than that determined by CD spectroscopy in the present study (12%) and elsewhere (15-16%). The high percentage of beta-sheet structure in apo B-100 supports the importance of such segments in maintaining the lipid-protein assembly in LDL. Polarized infrared spectroscopy indicates that the beta-sheet component of apo B-100 adopts a preferential orientation with respect to the phospholipid monolayer surrounding the LDL, whereas no such orientation is observed for the other secondary structure components.

Apolipoprotein B-100↗

PubMed1989

Separation and quantitation of free cholesterol and cholesteryl esters in a macrophage cell line by high-performance liquid chromatography.

A method for the direct high-performance liquid chromatographic (HPLC) determination of free cholesterol and the individual cholesteryl esters in cell culture experiments is described. The murine macrophage-like J774 cell line was loaded with cholesterol by incubation with low-density lipoproteins. After extraction of the cellular lipids with hexane-isopropanol (3:2, v/v), the cholesteryl esters were identified and quantified by isocratic HPLC. Unesterified cholesterol and its esters were eluted with acetonitrile-isopropanol (50:50, v/v) on a Zorbax ODS column within 25 min and detected at 210 nm. Cholesteryl heptadecanoate was used as an internal standard. The detection response is linear in the analytical range of interest; the overall coefficients of variation are less than 8% and the detection limit is between 50 and 150 ng. The results demonstrate that HPLC is suitable for the determination of cellular cholesteryl ester profiles and could usefully contribute to the understanding of the mechanism of foam cell formation during the development of atherosclerosis. This method can also be applied to all experimental systems involving the study of cholesteryl esters.

Animals↗

PubMed1989

The primary structure of human apolipoprotein A-IV.

Human apolipoprotein (apo) A-IV was purified from chylous ascites fluid. Proteolytic peptides produced by trypsin and Staphylococcus aureus V8 proteinase digestions were purified by high-performance liquid chromatography and sequenced. Human apoA-IV contains 376 amino acid residues. The peptide-derived sequence generally matches two previously reported DNA-derived amino acid sequences except for discrepancies in five positions. In order to examine these discrepancies further, one complete apoA-IV cDNA clone and another partial clone were sequenced. Comparison of all the available information indicates that the peptide-derived sequence reported here is accurate. Sequencing errors probably account for some of the discrepancies between the two primary sequences predicted by earlier nucleotide analyses. In certain positions, however, bona fide sequence heterogeneity or cloning artifact cannot be excluded.

Amino Acid Sequence↗

PubMed1989

Relationship between serum cholesteryl ester composition, dietary habits and coronary risk factors in middle-aged men.

Serum cholesteryl palmitate, oleate, linoleate and arachidonate were measured using high performance liquid chromatography in 134 men aged 45-64 years, randomly selected from the population of Ghent, Belgium. No relationship was observed between these cholesteryl esters, expressed as a percentage of their total, and age, serum cholesterol, arterial blood pressure, smoking habits and body mass index. Dietary habits were assessed using a 3-day diary method. No relationship was found between dietary habits and serum lipids. Highly significant correlations were observed between diet and the serum cholesteryl esters. In multivariate analyses dietary variables account for significant proportions of the variances of the serum cholesteryl esters. The results indicate that measurements of cholesteryl esters can be used to differentiate on the population level between subgroups with different dietary habits particularly with respect to fat consumption.

Age Factors↗

PubMed1989

Quantitation of plasma free cholesterol and cholesteryl esters by high performance liquid chromatography. Study of a normal population.

We describe a convenient method for the separation and quantitation of plasma free cholesterol and cholesteryl esters by high performance liquid chromatography (HPLC). After extraction of 100 microliters plasma with isopropanol the plasma cholesteryl esters were resolved on a Zorbax ODS reversed-phase column by isocratic elution with acetonitrile/isopropanol (50:50, v/v). Baseline separation of the plasma cholesteryl esters including the internal standard was obtained within a 25-min run. The intra- and interassay CV was less than 4%. The results obtained by HPLC show good agreement with enzymatic and gas-liquid chromatographic methods. High performance liquid chromatography provides a simple method for the quantitation of individual cholesteryl esters avoiding tedious chromatographic and derivatisation steps inherent to GLC. Our HPLC method was applied to the monitoring of plasma cholesteryl esters in a normal population and can also be used for the study of cholesteryl esters from lipid extracts of biological samples.

Adult↗

PubMed1989

Influence of development, estrogens, and food intake on apolipoprotein A-I, A-II, and E mRNA in rat liver and intestine.

UNLABELLED: The influence of development and ethinylestradiol (EE) on apolipoprotein (apo) A-I, A-II, and E mRNA in rat liver and intestine was studied by dot blot hybridization and Northern blot analysis. ApoA-I mRNA levels were maximal in the perinatal period and declined after day 15. An opposite trend was noted for the apoA-II mRNA levels, whereas apoE mRNA remained fairly constant. Liver apoA-I mRNA levels increased after ovariectomy (OVX). A further rise was observed when EE was given at 2000 micrograms/day. When the influence of OVX and EE was controlled for food intake by pair-feeding, OVX still increased hepatic apoA-I mRNA. The rise in liver apoA-I mRNA after EE, however, was no longer significant. Under the same conditions OVX slightly increased intestinal apoA-I mRNA. EE (2000 micrograms/day) decreased intestinal apoA-I mRNA to 80% of the pair-fed controls. Liver apoA-II mRNA levels did not change after OVX when the animals were fed ad libitum, but decreased slightly when the rats were pair-fed. EE caused a dose-dependent decrease in liver apoA-II mRNA, irrespective of food intake. None of these treatments caused any change in liver apoE mRNA levels. Serum apoA-I levels increased upon OVX, while serum apoE did not change. EE provoked a dose-dependent decrease of both apolipoproteins in serum. IN CONCLUSION: 1) Changes in food intake play an important role in the in vivo effects of estrogens on apolipoprotein mRNA levels. 2) The stimulatory effect of OVX on hepatic apoA-I mRNA as well as the inhibitory effect of EE on hepatic apoA-II mRNA are independent of food intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Lipoprotein(a) quantified by an enzyme-linked immunosorbent assay with monoclonal antibodies.

This new, sensitive, specific "sandwich"-type enzyme-linked immunosorbent assay (ELISA) for quantifying lipoprotein(a) [Lp(a)] in human serum and in ultracentrifugal lipoprotein fractions is based on use of a monoclonal antibody raised against apolipoprotein(a) as coating protein and a polyclonal antibody, raised against either apo B or against Lp(a) and conjugated with peroxidase, for detection of bound Lp(a). Mean intra- and interassay CVs for assay of 16 samples were 3.0% and 5.6%, respectively. Sample pretreatment with urea did not enhance Lp(a) immunoreactivity, and treatment with nonionic detergents decreased binding to the monoclonal antibody. Results correlated well (r = 0.99, n = 38) with those by radial immunodiffusion (RID). The ELISA assay, however, detects amounts corresponding to Lp(a) contents of 10 to 1000 mg/L in plasma samples diluted 1000-fold, compared with 100-500 mg/L for RID. For 92 normolipidemic subjects, the mean Lp(a) concentration was 120 (SD 130) mg/L. In patients undergoing coronary angiography, Lp(a) concentrations increased with the severity of the disease but were not correlated with either HDL cholesterol, triglycerides, apo A-I, or apo B, and only weakly with plasma cholesterol and apo A-II. These two correlations were even weaker in normal subjects, and only the correlation with total cholesterol was valid. Lp(a), measured at birth and at seven days and six months, steadily increased with age. This assay is well suited for measuring Lp(a) in plasma and in lipoprotein fractions and also for screening programs evaluating this significant genetic risk factor for the development of atherosclerosis.

Adult↗

PubMed1989

[Determinants of fat consumption in a general population].

In a random sample of the adult population (aged 25-64 yr) from Ghent and Charleroi in Belgium, dietary fat consumption was estimated using both qualitative and quantitative dietary methods. A fat consumption score (FCS) was constructed from the qualitative method and correlated significantly with the dietary P/S ratio calculated from the quantitative method. Differences in FCS were then studied using univariate and multivariate analyses. In both men and women FCS was higher in Charleroi than in Ghent. In men FCS was inversely related with educational level and with age and positively related with BMI. In women no significant correlations were observed. In the middle-aged group (aged 45-64 yr) significant correlations were found between FCS and the fatty acid composition of the cholesterol-esters, in particular with the linoleate/oleate ratio.

Adult↗

PubMed1988

Conformational analysis of lipid-associating proteins in a lipid environment.

Two major types of helical structures have been identified in lipid-associating proteins, being either amphipathic or transmembrane domains. A conformational analysis was carried out to characterize some of the properties of these helices. These calculations were performed both on isolated helices and in a lipid environment. According to the results of this analysis, the orientation of the line joining the hydrophobic and hydrophilic centers of the helix seems to determine the orientation of the helix at the lipid/water interface. The calculation of this parameter should be useful to discriminate between an amphipathic helix, parallel to the interface and a transmembrane helix orientated perpendicularly. The membrane-spanning helices are completely immersed in the phospholipid bilayer and their length corresponds to about the thickness of the hydrophobic core of the DPPC bilayer. The energy of interaction, expressed per phospholipid is significantly higher for the transmembrane compared to the amphipathic helices. For the membrane-spanning helices the mean energy of interaction is higher than the interaction energy between two phospholipids, while it is lower for most amphipathic helices. This might account for the stability of these protein-anchoring domains. This computer modeling approach should usefully complement the statistical analysis carried out on these helices, based on their hydrophobicity and hydrophobic moment. It represents a more refined analysis of the domains identified by the prediction techniques and stress the functional character of lipid-associating domains in membrane proteins as well as in soluble plasma lipoproteins.

1,2-Dipalmitoylphosphatidylcholine↗