Separation and quantitation of plasma lipoproteins by high-performance liquid chromatography.
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Adult↗
Biomedical subjects
M Rosseneu
Publications and source records attributed to M Rosseneu.
Isolation and characterization of lipoprotein profiles in newborns by density gradient ultracentrifugation.
Lipoproteins in newborn plasma were isolated from a minimal sample amount (0.3 ml) by a single-step ultracentrifugation in a density gradient, spanning the density range 1.02-1.20 g/ml. After 66 h ultracentrifugation in a swinging-bucket rotor, the content of the tube was eluted and collected in 0.4 ml fractions. Cholesterol and apoproteins AI, AII, and B were assayed in each fraction yielding both the distribution and composition of the very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL2, HDL3). Newborn plasma was characterized by a low amount of triglyceride-poor and cholesterol ester-rich VLDL and high content of HDL2 and HDL3. The VLDL and LDL concentrations increased drastically between 0 and 7 days together with the triglyceride content of the VLDL. At 30 days the lipid composition of VLDL was similar to that of adults, whereas the lipid/protein content remained low both in VLDL and LDL. The composition of HDL2 and HDL3 remained constant during this period, the percentage of HDL2 being higher in newborns than in adults. These compositional changes were reflected in the microviscosity of the lipoproteins, specially in the VLDL fraction.
Apoproteins↗
Some considerations of methodology and standardization of apolipoprotein B immunoassays.
Apolipoprotein B, the major protein component of very-low-density (VLDL) and low-density (LDL) lipoproteins in serum, is being widely measured in serum, to help assess risk of cardiovascular disease. Standardization has proven difficult because of the physical and immunochemical heterogeneity of LDL and VLDL, the masking of antigenic determinants of LDL and VLDL, the insolubility of apolipoprotein B, the instability of lipoprotein reference materials, and the multiplicity of analytical methods. Here we have examined the literature and collected current information from lipoprotein scientists to determine the problems--and the ways investigators have attempted to solve them--in preparation, storage, and use of standards and anti-apolipoprotein B sera, analytical procedures and reagents, and the nature of apolipoprotein B in serum. Standardization appears possible with development of stable reference materials that are applicable to the various analytical methods, controlled immunochemical reaction conditions, and establishment of a selected interim reference method for use as a point of reference.
Adult↗
Some considerations of methodology and standardization of apolipoprotein A-I immunoassays.
Apolipoprotein A-I (apo A-I) is the major protein of high-density lipoprotein. Compelling evidence suggests that measurement of concentrations of apo A-I in serum may be useful in predicting and assessing ischemic heart disease. The following review of methods of isolation, characterization, and assay of apo A-I has been developed to assess the possibility of standardizing apo A-I immunoassays. We consider the properties of apo A-I that have an effect on its quantification, such as self-association, polymorphism, and stability characteristics. We attempt to review critically the various methods presented, but more information about the physicochemical properties of the protein is required before definitive recommendations can be made.
Adult↗
Dissociation of apolipoprotein AI from apoprotein-lipid complexes and from high-density lipoproteins. A fluorescence study.
The dissociation of the apoprotein AI (apoAI) from the apoAI-dimyristoylglycerophosphocholine complex and from human high-density lipoproteins (HDL), was induced by incubation with guanidinium hydrochloride at concentrations between 0 M and 7 M. The kinetics and extent of denaturation were followed by monitoring both the fluidity of the lipid phase by fluorescence polarization measurements and the protein conformation by measuring the tryptophanyl fluorescence emission. The association with lipids protects apoAI against denaturation both in HDL and in the apoAI-phospholipid complex. The results of the kinetic and end-point measurements suggest that the denaturing effect of guanidine hydrochloride on the apoAI-lipid complex and on HDL is a two-step process. It involves the dissociation of the apoAI-phospholipid bond, as evidenced by fluidity measurements: this effect is maximal between 3 M and 4 M guanidine hydrochloride. The conformational change of the apoAI protein into a randomly coiled structure with the tryptophanyl residues exposed to the solvent is maximal between 4 M and 6 M guanidine hydrochloride. HDL and the apoAI-phospholipid complex have a closely similar behaviour towards denaturation by guanidine hydrochloride indicating that the phospholipid-apoAI association in HDL is primarily responsible for the stability of the lipoprotein molecule.
Apolipoprotein A-I↗
A screening procedure for dyslipoproteinemia in the newborn. Apoprotein quantitation on dried blood spots.
A new screening procedure for the detection of dyslipoproteinemia in the newborn is proposed, based on apoprotein quantitation. Blood is collected by heel-prick in infants 5--7 days after birth--when apoprotein and lipids have reached stable values--and adsorbed on filter paper. Blood spots are eluted with a detergent and apoprotein A-I and B are assayed by immunonephelometry. The apo A-I/B ratio is used as a screening parameter, given its high discriminative power between normals and individuals with cardiovascular disease. The quantitation of the apo A-I/B ratio in blood spots is independent of the volume of the blood spot and of the newborn hematocrit. In a follow-up study, plasma lipid and apoprotein concentrations were assayed in the infants with an apo A-I/B ratio lower than 1.0 at 5 days, and a family study was carried out. On screening 1500, 30 infants gave positive results at birth, and were controlled between 2 and 8 months. In eight families studied, six children had abnormally high cholesterol and apo B values and five children had abnormally low HDL cholesterol and apo A-I concentrations. This screening procedure is a new approach to the detection of familial dyslipoproteinemia in the newborn, as it is based on the quantitation of the apo A-I/B protein ratio, instead of cholesterol, LDL cholesterol or beta-lipoprotein quantitation. It enables a differentiation to be made between various forms of dyslipoproteinemia, leads to a better characterization of various diseases, and decreases the percentage of false positive cases.
Adult↗
Binding of beta-adrenoceptor blocking drugs to human serum albumin, to alpha 1-acid glycoprotein and to human serum.
The binding of 8 beta-adrenergic blocking drugs to human serum albumin, to alpha 1-acid glycoprotein and to serum from normal volunteers and from patients with rheumatoid arthritis was studied. Protein binding was determined in vitro using equilibrium dialysis of labelled drug at 25 degrees C. Oxprenolol and propranolol were highly bound to serum, alprenolol, pindolol and timolol to a lesser degree, and atenolol, metoprolol and sotalol were negligibly bound. For the five compounds which were appreciably bound, the mean binding was significantly higher in serum from patients with rheumatoid arthritis than in serum from normal volunteers. For those drugs, binding to alpha 1-acid glycoprotein was higher than to human serum albumin, and binding to a mixture of both proteins approached that to serum from healthy volunteers. For each of these drugs there was a strong correlation between the serum alpha 1-glycoprotein concentration and the percentage binding.
Adrenergic beta-Antagonists↗
Discriminative value of lipids and apoproteins in coronary heart disease.
Serum cholesterol, HDL cholesterol (HDL-C), and apoproteins, A1, A2 and B were determined in 70 male survivors of myocardial infarction and in an equal number of healthy controls, matched for age, sex and body mass index. In univariate analyses, the Apo B/Apo A1 ratio discriminated the best between cases and controls, giving a 72% exact classification. In a multivariate analysis, the Apo B/Apo A1 ratio, HDL-C and the Apo A2/Apo A1 ratio contributed independently to the discrimination of cases from controls while the overall exact classification was 82%. These promising results were comparable in younger and older subgroups. Thus, the determination of apoproteins yielded complementary information in this cross-sectional survey and warrants further study in a prospective setting.
Adult↗
Serum lipids and apolipoproteins A-I, A-II, and B in hyperthyroidism before and after treatment.
The serum concentrations of total cholesterol (TC), triglycerides (RG), high density lipoprotein-cholesterol (HDLc), low density lipoprotein-cholesterol (LDLc), and the apolipoproteins (apo) A-I, A-II, and B were measured in 33 hyperthyroid patients before and after treatment. The results were compared with those of healthy controls. Apo A-I, A-II, and B were assayed by immunonephelometry. The serum levels of TC (mean +/- SD, 167 +/- 36 mg/dl, HDLc (40.8 +/- 12 mg/dl), and LDLc (108 +/- 35 mg/dl) were decreased in the untreated hyperthyroid patients compared to both the values after treatment (TC: 215 +/- 54 mg/dl; P less than 0.001; HDLc: 52 +/- 14 mg/dl; P less than 0.001; LDLc: 146 +/- 47 mg/dl; P less than 0.001) and the control values (TC: 206 + 39 mg/dl; P less than 0.001; HDLc: 47.4 +/- 10 mg/dl; P les than 0.01; LDLc: 145 +/- 38 mg/dl; P less than 0.001). TG levels were not statistically different before and after treatment. The apo A-I concentrations (116 +/- 24 mg/dl) were lower before than after treatment (131 +/- 28 mg/dl; P less than 0.01), but they were not statistically different from those in the control group (115 +/- 19 mg/dl). The apo A-II levels were identical in all groups (before treatment, 35 +/- 7 mg/dl; after treatment, 37 +/- 9 mg/dl; control group, 36 +/- 9 mg/dl). The apo B levels were lower in the untreated hyperthyroid patients (86 +/- 23 mg/dl) compared to those in controls (103 +/- 19 mg/dl; P less than 0.001) and patients after therapy (103 +/- 25 mg/dl; P less than 0.001). The increase in HDLc relative to the major HDL apo A-I and A-II during treatment for hyperthyroidism was associated with changes in body weight. The apo A-I to apo A-II and LDLc to apo B ratios, however, were significantly lower before compared to those after treatment, when the influence of increasing body weight during therapy was accounted for. This study emphasizes the important regulating role of thyroid hormones on lipid and apolipoprotein metabolism.
Apolipoprotein A-I↗
Separation of lipoproteins in newborn plasma using an Airfuge ultracentrifuge.
The isolation of lipoprotein fractions in newborn plasma was performed by ultracentrifugation in an Air-driven ultracentrifuge (Airfuge). The purity and recovery of the fractions was checked by quantitation of the apo A1 and B proteins in the supernatant and infranatant fractions, and by gradient gel electrophoresis in 4-30% polyacrylamide gels. The influence of the duration of the ultracentrifugal run, the temperature and method for recovery of the fractions were tested. Under optimal conditions pure VLDL fractions could be isolated at d = 1.006 milligrams, whereas VLDL + LDL isolated at d = 1.063 milligrams were contaminated with HDL and other plasma proteins. Isolation of total lipoproteins at d = 1.21 milligrams enabled recovery of 75% of total plasma lipoproteins, however strongly contaminated by other plasma proteins. These results indicate that the recovery and purity of the lipoprotein fractions isolated in the Airfuge are not comparable to the results obtained in a conventional preparative ultracentrifuge.
Apolipoprotein A-I↗
Plasma apoproteins levels in chronic alcohol abuse.
The plasma lipid and apoprotein concentrations were monitored in a group of 12 patients with chronic alcohol abuse entering an abstinence program for 3 weeks. 6 of them had a normal liver function as expressed by the levels of liver enzymes gamma GT, GOT, GPT, while 6 had elevated plasma liver enzyme concentrations. None had evidence of either cirrhosis or alcohol hepatitis. Patients with abnormal liver enzymes had elevated HDL-cholesterol, apo AI and apo AII concentrations in plasma, with normal total cholesterol and apo 8 concentrations. In the group of patients with normal liver enzyme concentrations, the apoproteins and lipids did not significantly differ from the control group. In the course of the abstinence treatment a parallel decrease of apoproteins, HDL-cholesterol and liver enzyme concentrations was observed. The values normalized after 10-15 days. These data indicate that the effect of alcohol on the plasma apoprotein and lipids occurs mostly in the HDL fraction, that it correlates with the state of hepatic function and that it can be reversed by an abstinence treatment.
Adult↗
Standardization of immunoassays for the quantitation of plasma Apo B protein.
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Apolipoproteins↗
In vitro interaction of human HDL with human apolipoprotein A-II. Synthesis of apolipoprotein A-II-rich HDL.
The aim of this study was to define the specific affinity of human apolipoproteins A-I and A-II for HDL lipids and to investigate the possible transfer of apolipoproteins from the HDL molecule. For this purpose we incubated human HDL with increasing amounts of isolated apolipoprotein A-II. After incubation the reaction products were separated by gel chromatography and apolipoproteins A-I and A-II were quantified separately by immunonephelometry and HDL lipids by thin-layer chromatography. According to our results, apolipoprotein A-II progressively displaces apolipoprotein A-I to generate an HDL-like particle with identical lipid composition, hydrodynamic properties and lipid fluidity. These data indicate that apolipoprotein A-II is able to displace quantitatively apolipoprotein A-I from HDL in vitro, and that such a mechanism might contribute to the regulation of the HDL2 in equilibrium or formed from HDL3 distribution in plasma.
Apolipoprotein A-I↗
Effect of polyenephosphatidylcholine on cholesterol uptake by human high density lipoprotein.
The lipid and protein composition of human HDL was changed by incorporation of polyenephosphatidylcholine (PPC) into HDL in vitro. HDL with incorporated PPC (HDL-PPC) had a higher molar PC/apoprotein ratio than native HDL. PPC accounted for up to 50% of the PC fraction of HDL. The fluidity of HDL-PPC was higher than that of native HDL but lower than that of PPC liposomes. Zonal ultracentrifugation separated HDL-PPC into a major and a minor component. The AI/AII ratio of the major fraction was reduced compared with native HDL. The storage capacity of HDL-PPC and native HDL for cholesterol was studied by incubation of these fractions with [14]cholesterol-LDL. Significantly more cholesterol (55%) was taken up by HDL-PPC from LDL than by native HDL. The transfer of cholesterol from LDL to HDL in human serum was studied by an in vitro [14C]cholesterol distribution test. In this test the lipoproteins of serum were labelled with [14C]cholesterol. An analytical procedure was developed to quantify the transfer of cholesterol from LDL to HDL after addition of PC. The transfer depended on the fluidity and the dose of the PC fraction used as well as on the initial LDL + VLDL/HDL ratio and was independent of LCAT activity.
Carbon Radioisotopes↗
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.
Apolipoprotein A-I↗
Plasma apoprotein and lipid patterns in newborns: influence of nutritional factors.
In this study the plasma lipid and apoprotein concentrations have been assayed in 80 full-term newborns, at 0, 7 and 30 days of life, and the data have been analyzed as a function of the composition of the diet. The total cholesterol, HDL cholesterol, the apo A-I, A-II and B protein concentrations were followed in 4 groups of infants receiving respectively breast-feeding, adapted formulae I, II with a P/S ratio close to that of maternal milk and a formula III enriched with polyunsaturated fatty acids. After 7 and 30 days the infants receiving the adapted formulae I and II have plasma lipid and apoprotein values similar to those of the breast-fed infants indicating a parallel evolution of the lipids and apoproteins in the three groups. The lipid and apoprotein patterns were significantly different in the group of infants receiving a diet enriched with polyunsaturated fatty acids. The total and VLDL-LDL cholesterol and the apo B protein concentrations are significantly lower than in the breast-fed infants after 7 days, and these differences become more pronounced after 30 days. These results suggest that the fatty acid composition of the diet influences the lipid and lipoprotein synthesis in newborns, specially by decreasing the lipid and apoprotein concentrations of the VLDL-LDL fraction.
Apoproteins↗
Quantitative determination of human plasma apolipoprotein A-I by laser immunonephelometry.
A technical procedure is described for quantitation of human apolipoprotein A-I (apo A-I) in normal plasma or serum by immunonephelometry. Dilution of the plasma samples with 6 mol/L guanidine chloride ensures maximum exposure of the antigenic sites of the apoprotein and enables optimum quantitation of the apo A-I without requiring extraction with organic solvents. Similar data are obtained by this assay and with radioimmunoassay for normal subjects (1.2--1.5 g/L), and the results obtained on 31 patients are correlated with a coefficient of 0.92. The apo A-I values are correlated with values for plasma high-density lipoprotein cholesterol (r = 0.64). The interassay CV for immunonephelometry is about 7% and the standard curve is linear between 0.1 and 1.0 microgram of apo A-I per sample, corresponding to a 150-fold dilution of serum or plasma. The assay is applicable to plasma samples containing as much as 4 g of triglycerides per liter. At higher concentrations plasma delipidation is required.
Adult↗
Displacement of apo A-I by A-II in lipid-apoprotein complexes and human HDL.
The aim of this study was to define the specific affinity of human apo A-I and apo A-II for HDL lipids and to investigate the possible transfer of apoproteins from the HDL molecule. For this purpose we incubated apo A-I -- lipid complexes prepared "in vitro", as well as human HDL with increasing amounts of isolated apo A-II. After incubation the reaction products were separated by gradient ultracentrifugation and gel chromatography. The apoproteins were quantitated separately by immunonephelometry and the apo A-I content was monitored by measuring the intrinsic tryptophan fluorescence. These results suggest that apo A-II has a higher affinity than apo A-I for the lecithin-cholesterol vesicle and that 2 mol apo A-II are able to displace 1 mol apo A-I from the apo A-I lipid complexes. Analogous results were obtained with HDL where two mol apo A-II substitute to 1 mol apo A-I to yield en apo A-II - rich HDL with identical lipid composition, hydrodynamic properties and fluidity. Such a mechanism might contribute to the regulation of the HDL2 in equilibrium with HDL3 distribution in plasma.
Apolipoprotein A-I↗