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Biomedical subjects

G Schmitz

Publications and source records attributed to G Schmitz.

At least 271 records · Page 15Linked to original sources
PubMed1985

Characterization of very low density lipoproteins and intermediate density lipoproteins of normo- and hyperlipidemic apolipoprotein E-2 homozygotes.

Triglyceride-rich lipoproteins derived from ten normo- and hyperlipidemic apoE-2 homozygotes were analyzed for their composition, beta-VLDL content, and their ability to induce cholesteryl ester storage in macrophages. In six of these probands apoE sequence analysis revealed that the cysteine residues were at positions 112 and 158 of the amino acid sequence (Rall et al. 1983. J. Clin. Invest. 71: 1023-1031). ApoE-2 of these six and the other four patients was further analyzed by SDS electrophoresis to exclude the presence of apoE-2* (Rall et al. 1982. Proc. Natl. Acad. Sci. USA. 79: 4696-4700). The relative serum concentrations of free and esterified cholesterol transported in the d less than 1.006 g/ml and d 1.006-1.019 g/ml lipoproteins of the apoE-2 homozygotes was significantly higher as compared to controls. Compositional analysis of these lipoproteins revealed a relative reduction of triglycerides and a relative increase of cholesteryl esters as compared to controls. In most patients, with increasing serum triglyceride levels the cholesteryl ester concentration increased in d less than 1.006 g/ml and d 1.006-1.019 g/ml lipoproteins. However, in three patients with a low content of beta-VLDL, the increase in the d less than 1.006 g/ml fraction cholesterol was mostly due to free cholesterol and not due to cholesteryl esters. The degree of the macrophage cholesteryl ester accumulation induced by d less than 1.006 g/ml lipoproteins was mostly dependent on the concentration of the beta-migrating fraction (beta-VLDL). The amount of beta-VLDL and pre-beta-VLDL contained in the d less than 1.006 g/ml fraction was determined densitometrically after electrophoretic separation. It could be demonstrated that the beta-VLDL content in the d less than 1.006 g/ml fraction of the apoE-2 homozygous patients was largely independent of serum triglyceride and serum cholesterol levels. When macrophages were incubated with the IDL fraction (d 1.006-1.019 g/ml) from the apoE-2 patients, no significant increase in cellular cholesteryl esters above control levels was observed. Studies with purified lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) clearly revealed that both enzymes interacted with apoE-2 VLDL (binding, hydrolysis) to a lesser degree compared to control preparations. However, the apoE-2 VLDL preparations containing a low content of beta-VLDL were better substrates for LPL and HTGL than those containing a high beta-VLDL content. It is concluded from our studies that the plasma beta-VLDL content in apoE-2 homozygotes is a major determinant for cholesteryl ester accumulation in macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1984

A quantitative densitometric method for the rapid separation and quantitation of the major tissue and lipoprotein lipids by high-performance thin-layer chromatography. I. Sample preparation, chromatography, and densitometry.

A rapid method for the separation and quantitation of the major lipids of tissues and lipoproteins by automated high-performance thin-layer chromatography is presented. Solvent systems for one-dimensional separation of neutral lipids, of cholesteryl esters, and of phospholipids are described. Separated lipids are measured following treatment with methanolic sulphuric acid containing manganese chloride and scanned in fluorescence or absorption mode. Absolute quantitation is obtained by the use of an internal standard and by references to standards for each lipid run on the same plates as samples. The method described here is particularly suitable for the rapid quantitation of small amounts of lipid (0.01-0.02 nmol per sample), for example in tissue culture studies; 100 micrograms of fibroblast or macrophage protein are sufficient for complete lipid analysis. The coefficients of variation due to the sample preparation, application to the plates and densitometry are in the range 7.2-9.1%. The method was compared with enzymatic determinations for cholesterol and gave correlation coefficients of 0.95 for total cholesterol and 0.91 for unesterified cholesterol. Phospholipid estimation was compared with large-plate thin-layer chromatography and phosphorus analysis and gave correlation coefficients of 0.90 for phosphatidylcholine and 0.89 for sphingomyelin.

Animals↗
PubMed1984

A quantitative densitometric method for the rapid separation and quantitation of the major lipids of tissues and lipoproteins by high-performance thin-layer chromatography. II. Reduction of the densitometric data.

A BASIC program is described for acquisition of data and data reduction for an automated densitometric system for quantitation of lipids separated by high-performance thin-layer chromatography. The program allows calculation of mass of samples from log/log calibration curves computed from standards. The calculated masses are reported as nmol/volume or nmol/mg protein. The program contains a flexible dialog system which permits its use for a variety of applications in addition to the system described for quantitation of lipids.

Autoanalysis↗
PubMed1984

Topography and dynamics of receptors for acetylated and malondialdehyde-modified low-density lipoprotein in the plasma membrane of mouse peritoneal macrophages as visualized by colloidal gold in conjunction with surface replicas.

The topography and dynamics of receptors for acetylated (acetyl) and malondialdehyde-modified (MDA) low-density lipoprotein (LDL) in the plasma membrane of cultured mouse peritoneal macrophages were investigated using a new technique. Modified LDL labeled with gold particles was used to visualize LDL receptors in the plane of the plasma membrane in platinum-carbon surface replicas of critical point-dried cells. It was found that the native distribution of unoccupied acetyl-LDL receptors is diffuse, whereas unoccupied MDA-LDL receptors are preclustered in the plasma membrane. Competition and double labeling experiments suggest the existence of two distinct classes of receptor sites for acetyl-LDL and MDA-LDL.

Acetylation↗
PubMed1984

Lipid binding properties of the Tangier apolipoprotein A-I and its isoproteins.

The apolipoprotein A-I was isolated from the plasma of normal individuals and of three homozygous patients with Tangier disease by immunoprecipitation. The apoA-I isoforms were further fractionated by isofocusing on polyacrylamide gels. The physicochemical behavior of normal and Tangier apoA-I and of the isoproteins-2 and -4 was studied by monitoring the tryptophanyl fluorescence emission as a function of temperature, pH, and under exposure to guanidinium (guanidine) hydrochloride (GdmCl). Lipid-apoprotein complexes were generated by incubation with dimyristoylphosphatidylcholine and isolated by density gradient ultracentrifugation. Our results show that normal apoA-I and its isoprotein-4 associate with lipids to yield a complex containing 150-200 mol lecithin/mol apoA-I. The isoprotein-2 of normal apoA-I and the isoprotein-4 of Tangier apoA-I generate lipid-rich complexes with lecithin, while the isoprotein-2 of Tangier apoA-I shows only a limited association with lipids. ApoA-I normal and Tangier and their isoproteins-4 undergo a structural transition around 45 degrees C, which is not observed in the lecithin-apoA-I complexes. This transition is accompanied by an increased exposure of the tryptophanyl residues to the solvent. This transition was observed for the isoprotein-2 of apoA-I Tangier both in its lipid-free form and in the presence of lecithin. The pH denaturation of apoA-I and of the isoprotein-4 between pH 9 and 13 and between pH 7 and 2 is accompanied by a similar conformational transition. The transition occurs around pH 10.8 for the native apoproteins and is shifted towards respectively higher and lower pH's as result of the protective action of lipid binding on the protein conformation. Such an effect was not observed with the isoprotein-2 of apoA-I Tangier which is denatured at lower pH's both in its native form and in a lipid-protein mixture. Finally the denaturation of apoA-I by GdmCl indicates that apoA-I normal and Tangier undergo structural changes around 1 M GdmCl, whereas the apoA-I-Tangier-lecithin complex is more susceptible to denaturation than the complex with apoA-I normal. These data suggest that the apoA-I normal and Tangier and their isoproteins-4 are able to associate with lipids although the association between apoA-I Tangier with lecithin is weaker than that of apoA-I normal. The isoprotein-2 of normal apoA-I associates to a greater extent with lipids than the isoprotein-2 of Tangier apoA-I, whose structure differs from that of the isoprotein-4.(ABSTRACT TRUNCATED AT 400 WORDS)

Apolipoprotein A-I↗
PubMed1984

Acute influence of smoking on plasma lipoproteins.

This study was designed to investigate acute effects of smoking on plasma lipoproteins. Twelve fasting smokers inhaled the smoke of two cigarettes. Blood samples were taken before and 30 min after smoking. Triglycerides decreased in plasma from 136.8 +/- 39.8 to 108.0 +/- 34.9, in VLDL from 79.8 +/- 31.4 to 58.4 +/- 28.5 mg%. Cholesterol fell in HDL3 from 29.9 +/- 5.0 to 26.6 +/- 5.8 mg%. Determination of HDL-cholesterol with the phosphotungstic acid precipitation method revealed a reduction from 45.7 +/- 10.2 to 40.3 +/- 11.1 mg% after smoking. All C-apoproteins decreased in VLDL as an entity. Apoprotein A-I was diminished in plasma from 137.5 +/- 23.1 to 118.2 +/- 25.4, and in HDL3, from 97.1 +/- 12.1 to 85.0 +/- 13.1, apoprotein A-II in plasma from 28.6 +/- 8.9 to 24.7 +/- 6.9 and in HDL3 from 21.1 +/- 8.4 to 17.4 +/- 5.7 mg%. Zonal ultracentrifugation of D less than 1.063 g/ml lipoproteins confirmed the influence of smoking on VLDL. In LDL, an increase in cholesterol esters, mainly cholesterol linoleate, from 57.3 +/- 30.2 to 67.4 +/- 25.9 mg% was observed after smoking. The HDL2 subclass was shifted toward a slightly lighter hydrated density, but the major effect of smoking was seen in HDL3. Total cholesterol decreased from 30.5 +/- 7.3 to 22.3 +/- 6.6 mg%, due to a fall of all major cholesterol esters from 23.7 +/- 7.2 to 15.8 +/- 6.9 mg%. HDL3-phospholipids were reduced from 74.9 +/- 37.0 to 46.0 +/- 23.9 mg%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1984

Apolipoprotein E polymorphism and hyperlipidemia.

We tested apolipoprotein E phenotypes in 1557 normolipidemic factory workers and 822 hyperlipidemic hospital patients. We distinguished six different apolipoprotein E phenotypes and determined their frequencies in normolipidemia (factory workers), hypertriglyceridemia, hypercholesterolemia, and mixed hyperlipidemia. For the three homozygous phenotypes E3/3, E4/4, and E2/2, the percentage distribution in the normolipidemic group was 62.2%, 2.2%, and 0.9%, respectively; for the three heterozygous phenotypes E4/3, E3/2, and E4/2, we determined frequencies of 19.9%, 11.7%, and 2.9%, respectively. A higher prevalence of E2/2 homozygosity was observed in hypertriglyceridemic persons (2.5%) and persons affected by mixed hyperlipidemia (5.0%). E4/4 homozygosity occurred more often among hypercholesterolemic patients (5.0%) than normolipidemic persons (2.2%). These data suggest that E2/2 homozygosity and E4/4 homozygosity both predispose to hyperlipidemia. Patients affected by mixed hyperlipidemia should be investigated for their apolipoprotein E polymorphism because of the possible linkage of apolipoprotein E2/2 homozygosity, hyperlipidemia, and atherosclerosis.

Adult↗
PubMed1983

Tangier disease: defective recombination of a specific Tangier apolipoprotein A-I isoform (pro-apo A-i) with high density lipoproteins.

Isoforms of apolipoprotein A-I (apo A-I) from subjects with Tangier disease were characterized, and their ability to recombine with normal high density lipoproteins (HDL) was studied. In contrast to normal serum, in which isoprotein 4 is the dominant species [79 +/- 1.8% (mean +/- SD)], the Tangier serum contained much less total apo A-I (approximately equal to 1% of that in normal serum), and isoproteins 2 and 4 were present in roughly equivalent amounts (35.3 +/- 2.5% and 42.7 +/- 3.6%, respectively). The Tangier isoprotein 2 was shown to correspond to pro-apo A-I, having a six-amino acid amino-terminal extension with the sequence: Arg-His-Phe-Trp-Gln-Gln-. The Tangier isoprotein 4 had the same amino-terminal sequence as normal circulating plasma apo A-I. Its association with normal HDL (70%) was similar to the association of normal apo A-I with HDL (80-90%) in recombination experiments. In marked contrast to this behavior, very little (less than 10%) of Tangier isoprotein 2 (pro-apo A-I) associated with HDL in recombination experiments. These results suggest that the underlying defect in Tangier disease may be a faulty conversion of pro-apo A-I to mature apo A-I, either due to a defect in the converting enzyme activity or to a further specific structural defect in Tangier apo A-I. The failure of Tangier pro-apo A-I to associate with HDL may be at least partially responsible for the HDL deficiency in Tangier subjects.

Amino Acid Sequence↗
PubMed1983

Isoproteins of human apolipoprotein A-II: isolation and characterization.

In human serum, polymorphism of apoA-II predominantly in HDL3 could be demonstrated. HDL3-apoA-II was composed of four isoproteins, each with a molecular weight of 8600 (reduced form) and identical immunological properties. The isoproteins are designated apoA-II-1 (pI 5.16), apoA-II-2 (pI 4.89) corresponding to the already known apoA-II monomer band, apoA-II-3 (pI 4.58), and apoA-II-4 (pI 4.31). The amino acid compositions of the A-II isoproteins were virtually identical with the published data for apoA-II. Treatment with acid phosphatase, alkaline phosphatase, or neuraminidase before electrophoresis did not alter the apoA-II pattern. The apoA-II isoprotein pattern was studied in ten male and ten female normolipidemic volunteers, in two patients with Tangier disease, and in three patients with abetalipoproteinemia. The isoelectric focusing patterns of apoA-II appeared virtually identical in all subjects. However, in Tangier disease, due to the low apo-A-II concentration, only apoA-II-1 and apoA-II-2 were detectable, and in abetalipoproteinemia a different relative distribution pattern of the individual isoforms was found as compared to normal HDL3. Our studies indicate that apoA-II, similar to apoA-I, exists in several isoforms. The relationship of these isoforms to each other is at present unclear. They may originate from relatively basic isoproteins that are modified in charge by post-translational processes such as proteolytic cleavage, sequential deamidation, or other mechanisms.

Abetalipoproteinemia↗
PubMed1983

Quantification of high-density-lipoprotein cholesterol by precipitation with phosphotungstic acid/MgCl2.

We evaluated the use of a modified phosphotungstic acid/MgCl2 precipitation procedure for the precipitation of apolipoprotein B-containing lipoproteins. Precipitation of these lipoproteins [very-low- and low-density lipoproteins, and lipoprotein (a)] is complete, with negligible coprecipitation of high-density lipoprotein subfractions (HDL1, HDL2, HDL3), even in hypertriglyceridemic sera. In comparison with ultracentrifugation, the precipitation method yields, on the average, values that are 0.17 mmol/L lower for cholesterol values but almost identical for apolipoprotein A-I and phosphatidylcholine. Looking for delta 3,5-cholestadiene formed from cholesterol in the precipitation residue, we used "high-performance" liquid chromatography and "high-performance" thin-layer chromatography and found none.

Apolipoproteins↗
PubMed1983

Densitometry of phosphatidylcholine and sphingomyelin in high-density lipoproteins.

We describe the quantitative densitometric determination of phosphatidylcholine (PC) and sphingomyelin (SP) in human serum after precipitation with phosphotungstic acid/MgCl2 and use of thin-layer chromatography. After development, chromatographic plates were charred with methanolic sulfuric acid and MnCl2 and scanned by direct reflectance densitometry in an automated densitometric system interfaced to a basic programmable computing integrator. The method is sensitive enough to detect abnormally low concentrations of PC and SP in high-density lipoproteins. The accuracy of the method was tested either with the Bartlett phosphorus assay or with enzymatic methods for PC and SP; correlations of the described method with the enzymatic determinations were r = 0.93 and 0.88, respectively. Day-to-day precision (CV) for the phospholipid determination was 8.6% for PC and 12.2% for SP. The major advantage of this inexpensive technique is that native plasma or serum or the serum supernate after precipitation can be used without prior delipidation. With this technique serum high-density lipoproteins had PC values of 1.08 (SD 0.32) mmol/L in men (n = 158) and 1.12 (SD 0.37) mmol/L in women (n = 192); similarly, SP values were 0.23 (SD 0.07) mmol/L in the men and 0.23 (SD 0.08) mmol/L in the women. The differences by sex are not significant.

Adult↗
PubMed1982

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↗
PubMed1982

The role of lecithin: cholesterol acyltransferase in high density lipoprotein3/high density lipoprotein2 interconversion.

Serum was incubated in vitro with and without inhibition of lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43). High density lipoprotein2 (HDL2) and high density lipoprotein3 (HDL3) were separated by zonal ultracentrifugation and analysed for lipid and apoprotein contents. The incubation of fresh sera resulted in a time-dependent decrease in HDL3 and an increase in HDL2. At the end of 24 h incubation HDL3 disappeared completely and the HDL2 peak had reached its maximum. The newly formed HDL2 was relatively enriched in total protein (apoprotein A-I, C-apoproteins) and cholesteryl esters, and depleted in phosphatidylcholine. Its migration in polyacrylamide gel electrophoresis was identical with HDL2 contained in fresh serum or HDL2 isolated from serum by zonal ultracentrifugation. The generated HDL2 particles exhibited the same electron microscopical characteristics as reference HDL2 samples prior to incubation. Addition of Ellman's reagent to the incubation mixture or heat inactivation of the samples prior to incubation resulted in a complete inhibition of HDL3/HDL2 interconversion, whereas addition of 1 mol/l NaCl had no detectable influence. There was also a substantial increase in HDL2 when VLDL-deficient serum was incubated at 37 degrees C. Similarly, in fresh serum from a patient affected with familial lipoprotein lipase deficiency, HDL3 was completely converted to HDL2. Our experiments demonstrate that LCAT promotes HDL3/HDL2 interconversion in native serum irrespective of the presence or absence of triglyceride-rich lipoproteins and lipoprotein lipase.

Adult↗
PubMed1982

Isolation of human serum HDL1 by zonal ultracentrifugation.

High density lipoprotein subfraction-1 (HDL(1)) is thought to interact with the high-affinity apoprotein B, E receptors of peripheral cells and may act as a modulator of LDL binding and uptake. In the present study the concentration and composition of HDL(1) in normal and hypercholesterolemic sera were studied using zonal ultracentrifugation. To permit separation of the HDL(1) from VLDL, LDL, and Lp(a), the apoB-containing lipoproteins were first precipitated from serum using the phosphotungstic acid/magnesium chloride (PTA/MgCl(2)) method after which the supernatant fraction was subjected to zonal ultracentrifugation. It could be demonstrated that following PTA/MgCl(2) precipitation HDL(1) floats as a single peak at d 1.08-1.09 g/ml (NaBr) and is sufficiently separated from high density lipoprotein-2 (HDL(2)) and high density lipoprotein-3 (HDL(3)). The HDL(2)/HDL(3) subfraction pattern was not affected by the precipitation method. As previously described, in vitro incubation of serum leads to the LCAT-dependent interconversion of HDL(3) or HDL(2). Using the technique described here, it was discovered that a simultaneous elevation of HDL(1) occurred. This increase in HDL(1) concentration could not be observed when LCAT was inhibited by heat inactivation or addition of Ellman's reagent. In normal fresh serum only a small HDL(1) peak could be detected, but in patients with familial hypercholesterolemia (apoB, E receptor deficiency) HDL(1) was elevated five to tenfold compared to normal values and further increased in concentration upon incubation of serum. On the other hand, in sera of patients with familial HDL deficiency (Tangier disease), HDL(1) was undetectable. Analysis of the HDL fractions in serum of a patient with abetalipoproteinemia revealed that following in vitro incubation there was formation of HDL(1) despite the lack of apoprotein B-containing lipoproteins. These data support the concept that HDL(1) formation occurs during LCAT-mediated HDL(3)/HDL(2) interconversion in vitro.-Schmitz, G., and G. Assmann. Isolation of human serum HDL(1) by zonal ultracentrifugation.

Apolipoproteins↗
PubMed1981

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↗
PubMed1980

Excretion of a protease by Serratia marcescens.

Excretion of an extracellular protease of Serratia marcescens ATCC 25419 occurred during logarithmic growth and was highest (per cell) when cultures reached the stationary growth phase. Production of the extracellular protease was induced by leucine or casein in minimal medium or by growth in tryptone-yeast medium. In the late stationary phase an intracellular protease activity accumulated which was also observed in mutants with very low extracellular protease activity. The excreted protease was the dominant protein in the growth medium. The protease was purified to homogeneity by column chromatography on Bio-Gel P-100 and on DEAE-cellulose. Quantitative amino acid analysis revealed the absence of sulfur-containing amino acids. The enzyme consists of one polypeptide chain. A molecular weiht of 51,000 and 55,000 was estimated using polyacrylamide gel electrophoresis and chromatography on Bio-Gel P-100 respectively. The enzyme cleaved only N-alpha-benzoyl-DL-lysine-and-arginine-nitroanilides but not the corresponding leucine or tyrosine derivatives nor a set of di- and tripeptides.

Amino Acids↗