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Biomedical subjects

G M Kostner

Publications and source records attributed to G M Kostner.

At least 127 records · Page 7Linked to original sources
PubMed1994

[MED-PED: make early diagnosis--prevent early death].

The goal of MED-PED, a worldwide program including USA and many countries of Europe, Asia and Australia aims at diagnosing at the DNA level familial hypercholesterolemia (FH). There are currently 2 genetic defects under consideration: the LDL-R defect and familial Apo-B-100 defect (FDB). Whereas currently more than 160 mutations for the LDL-receptor defect are known, which can hamper the straight foreward characterization at the DNA level, FDB is caused by a single point mutation which may be easily characterized by routine methods. Austria participates since January 1st in MED-PED with subcenters in many different regions, which are listed in this article. We expect from the MED-PED program not only an optimation of health care in our country, with respect to preventive medicine, but also as studies from the US demonstrate, a positive cost effectiveness may be reached by this program. MED-PED in Austria is just at the beginning and in a rather preliminary stage, but we hope that after finding sponsors for covering the actual costs, this project will go on to a more advanced stage.

Apolipoprotein B-100↗
PubMed1993

Immunoquantification of lipoprotein(a): comparison of nephelometry with electroimmunodiffusion.

A new fully automated nephelometric immunoassay for lipoprotein(a) quantification in human serum was evaluated using the Behring Nephelometer Analyzer. The assay exhibited a good linearity in the concentration range of 110-1,770 mg/l; at higher concentrations, samples were automatically diluted by a factor of 4. The method is simple, robust, and shows an excellent stability of the calibration curve over several weeks. Intra-assay and day-to-day coefficients of variation were 2% and 4.5%, respectively. The method correlated well with electroimmunodiffusion (r = 0.977; n = 123; P = 0.0001). Unspecific turbidity as expressed by an elevated blank value occurred in 3% of all freshly measured samples (n = 392). Storage of the samples for 1 week at 4 degrees C had no significant influence on the results. Frozen sera, on the other hand, cannot be assayed by this method. We believe that this assay is well suited for use in clinical routine work.

Calibration↗
PubMed1993

A novel missense mutation in the gene for lipoprotein lipase resulting in a highly conservative amino acid substitution (Asp180-->Glu) causes familial chylomicronemia (type I hyperlipoproteinemia).

A previously undescribed single missense mutation (C-->G) was detected within exon 5 of the LPL gene in two members of an Italian family affected with type I hyperlipoproteinemia. This mutation causes a highly conservative amino acid replacement (Asp-->Glu) at position 180 of the mature LPL protein resulting in a virtual absence of LPL enzyme activity and LPL enzyme mass in postheparin plasma. Adipose tissue mRNA concentrations and mRNA sizes were not affected. Both patients were homozygous for the mutation, whereas the parents were heterozygous. Comparison of the expression of the mutated cDNA and the wildtype cDNA in cos-7 cells revealed proper transcription and translation of the mutated clone into an immunologically detectable protein. The mutated LPL protein was secreted from the cells in a manner similar to that of wild-type LPL and bound to heparin-Sepharose with identical properties. However, the mutated enzyme, in contrast to wildtype LPL, exhibited no detectable lipolytic activity against a triglyceride substrate. Our results demonstrate that even a highly conservative amino acid replacement outside the proposed active site of LPL is incompatible with proper enzyme function.

Amino Acid Sequence↗
PubMed1993

In vivo kinetics of lipoprotein(a) in homozygous Watanabe heritable hyperlipidaemic rabbits.

In vivo kinetics of lipoprotein(a) [Lp(a)] were investigated in homozygous Watanabe heritable hyperlipidaemic (WHHL) rabbits (an animal model of familial hypercholesterolemia (FH)), and in normolipidemic Japanese White rabbits (controls). 125I-labelled Lp(a) and 131I-labelled LDL were simultaneously injected intravenously. Blood samples were then taken periodically. Kinetic parameters were calculated from the plasma radioactivity decay curves. The fractional catabolic rates (FCRs) of both Lp(a) and LDL (1.355 +/- 0.189 pools per day and 1.278 +/- 0.397 pools per day, respectively) in the WHHL rabbits were significantly (P < 0.005) smaller than those in the control rabbits (2.008 +/- 0.083 pools per day and 2.855 +/- 0.759 pools per day, respectively). In WHHL rabbits, the FCRs of Lp(a) and LDL were similar. However, in control rabbits, the FCR of Lp(a) was significantly (P < 0.01) smaller than that of LDL. In WHHL rabbit organs, the mean ratio of 125I-Lp(a): 131I-LDL, 48 h after injection, normalized to the corresponding isotope ratio in plasma, were 1.525, 1.020, 1.819 and 1.967, in liver, kidney, spleen and bile, respectively. These values were significantly higher than the corresponding values in control rabbits (0.590, 0.677, 0.862 and 0.766, respectively). Our data strongly suggest that Lp(a) clearance is not entirely dependent upon LDL receptors and may be mediated by some other mechanisms.

Animals↗
PubMed1993

Interaction of Lp(a) and of apo(a) with liver cells.

Lipoprotein(a) (Lp[a]) is a lipoprotein of high atherogenicity with unknown function. Although it binds in vitro to the low-density lipoprotein (LDL) receptor, it is not clear whether this mechanism also operates in vivo. We studied the interaction of Lp(a) and of apoprotein(a) (apo[a]) with hepatoma cells (HepG2 and Hep3B) with the following results. (1) HepG2 cells exhibited saturable high-affinity binding of LDLs, whereas the majority of Lp(a) binding was of low affinity and nonsaturable. Preincubation of HepG2 cells with LDL markedly reduced cholesterol biosynthesis, but Lp(a) had a much lower effect. (2) When HepG2 cells were preincubated for 48 to 72 hours with Lp(a) or apo(a), 125I-LDL binding was increased by a factor of > 2. During this time, up to approximately 1 microgram of apo(a) per 1 milligram cell protein was found to be cell associated in an undegraded form. Monoclonal antibodies against the LDL receptor did not prevent the increase in LDL binding stimulated by apo(a). (3) Coincubation with LDL caused a significant increase of Lp(a) degradation by HepG2 cells that was probably caused by an increase of Lp(a) uptake in a "hitchhiking"-like process.

Apolipoproteins A↗
PubMed1993

Determination of lipoprotein(a): evaluation of three methods.

Lipoprotein(a) (Lp(a)) is a strong independent risk factor for premature atherosclerosis. Structurally, Lp(a) closely resembles LDL. Its protein moiety contains apolipoprotein B-100 and apolipoprotein(a). We evaluated two commercial enzyme immunoassays (EIAs) and an immunoradiometric assay (IRMA) for Lp(a). The three assays differed in their design and they used different antibodies. In the immunoradiometric assay, two different monoclonal antibodies were used. In the first EIA, monoclonal anti-apolipoprotein(a) was bound to the solid phase and Lp(a) was detected with polyclonal anti-apolipoprotein B (Lp(a):B-EIA). In the second EIA, polyclonal anti-apolipoprotein(a) was used as capturing antibody and as detecting antibody (apo(a)-EIA). Ninety three plasma samples were assayed for Lp(a) with the three methods. The best correlation was obtained between the IRMA and the Lp(a):B-EIA (r = 0.971). Correlations between the apo(a)-EIA on the one hand and the IRMA or the Lp(a):B-EIA on the other hand were 0.889 and 0.836, respectively. The methods significantly differed in their calibration. This resulted in different mean Lp(a) concentrations. When tested against purified Lp(a), the apo(a)-EIA appeared accurately calibrated, whereas the IRMA and the Lp(a):B-EIA overestimated Lp(a) by approximately twofold. In the Lp(a):B-EIA, the detecting antibody is directed against apolipoprotein B. The Lp(a):B-EIA is, therefore, not affected by apolipoprotein(a) size polymorphism. This allows expression of the concentration of Lp(a):B complexes on a molar basis. In contrast, the polyclonal antibody-based apo(a)-EIA measures the concentration of apolipoprotein(a) antigen, and may, therefore, be susceptible to inter- and intra-individual polydispersity of apolipoprotein(a) and Lp(a) particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

Organization of phosphatidylcholine and sphingomyelin in the surface monolayer of low density lipoprotein and lipoprotein(a) as determined by time-resolved fluorometry.

Fluorescent analogs of phosphatidylcholine (PC) and sphingomyelin (SM) labeled with diphenylhexatrienylpropionic acid (DPH) were prepared and incorporated into the surface layer of human low density lipoprotein (LDL) and lipoprotein(a) (Lp(a)). Fluorescence anisotropy measurements of DPH-PC and DPH-SM in both lipoprotein classes were carried out at different temperatures ranging from 20 to 37 degrees C. DPH-PC as well as DPH-SM were shown to reside in more rigid domains in Lp(a) than in LDL according to higher anisotropy values in Lp(a). In both LDL and Lp(a), DPH-PC experienced a more rigid environment than DPH-SM, suggesting different environments of PC and SM in the surface shell of the lipoproteins. Fluorescence lifetimes of the labeled lipoproteins were determined by phase and modulation fluorometry. We found bimodal Lorentzian distributions for the decay times of DPH-PC and DPH-SM in LDL and Lp(a). Lifetime distribution centers for labeled lipids were very similar except for DPH-PC in Lp(a) which was shifted to longer lifetimes, suggesting a less polar environment of PC in Lp(a) than in LDL. The distributional width of DPH-PC in Lp(a) was broader than in LDL. Accordingly, phosphatidylcholine must be localized in a more homogeneous environment in LDL as compared with Lp(a). On the other hand, no difference in distributional widths was observed for DPH-SM in both lipoproteins, showing that SM organization in Lp(a) is unaffected by apo(a). From the obtained fluorescence data we propose that apoproteins discriminate between the choline phospholipids and preferentially associate with phosphatidylcholine. This effect is enhanced in Lp(a) due to the presence of apolipoprotein(a).

Fluorescence Polarization↗
PubMed1992

High-density lipoprotein antagonizes the inhibitory effects of oxidized low-density lipoprotein and lysolecithin on soluble guanylyl cyclase.

Oxidatively modified LDL (LDLox) reduces the response of soluble guanylyl cyclase to nitrovasodilators. We now demonstrate that this desensitization can be antagonized by HDL. Similar to its protective effect against LDLox, HDL also inhibited the lysolecithin-induced desensitization of soluble guanylyl cyclase. Since the lysolecithin content of LDLox correlated with the amount of lysolecithin necessary to diminish stimulation of soluble guanylyl cyclase, our data support the hypothesis that lysolecithin may be responsible for the inhibitory effect of LDLox on smooth muscle relaxation and provide evidence that the antagonistic effect of HDL against desensitization of soluble guanylyl cyclase by atherogenic compounds could be responsible for the protective role of HDL in atherosclerosis.

Animals↗
PubMed1991

Sulfhydryl-selective fluorescence labeling of lipoprotein(a) reveals evidence for one single disulfide linkage between apoproteins(a) and B-100.

Human lipoprotein(a) and low-density lipoprotein were labeled with two different sulfhydryl-selective fluorescence markers. The hydrophilic fluorophore lucifer yellow iodoacetamide and the apolar compound 6-acryloyl-2-(dimethylamino)naphthalene were used to derivatize free -SH groups in the lipoproteins. Three sulfhydryls could be detected in low-density lipoprotein, whereas only two cysteines were available in lipoprotein(a). One of the three -SH groups in low-density lipoprotein was shown to be located in close proximity to the particle surface. We suggest that this surface-exposed cysteine of apoprotein B-100 serves as a component for the disulfide linkage to apoprotein(a) in lipoprotein(a).

2-Naphthylamine↗
PubMed1991

Lipoprotein(a) mediates high affinity low density lipoprotein association to receptor negative fibroblasts.

Lipoprotein(a) (Lp(a)) is an acute phase protein with unknown function. Lp(a) binds to low density lipoprotein (LDL) receptors, as well as to plasminogen (Plg) receptors. Preincubation of normal human skin fibroblasts with Lp(a) or with apo(a) cause a severalfold increase of LDL binding. Plg and kringle-4 of Plg have no effect. LDL receptor-negative fibroblasts respond upon preincubation with apo(a) with high affinity binding of LDL with Kd values that are almost identical with those of LDL binding to the LDL receptor. Incubation of apo(a)-pretreated fibroblasts with anti-apo(a) completely abolishes the increment of LDL binding. The high affinity LDL binding to LDL receptor-negative fibroblasts could be dissociated by approximately 80 and 54% with 5 mg/ml proline and 30 mg/ml NaCl, respectively, but not with dextran sulfate. The Lp(a)- and apo(a)-triggered LDL binding to fibroblasts have no effect on LDL internalization. These findings may reflect a key function in the role as an acute phase protein and may be relevant to the high atherogeneicity of Lp(a).

Apolipoproteins↗
PubMed1991

Oxidation of lipoprotein Lp(a). A comparison with low-density lipoproteins.

Aimed at identifying possible mechanisms of the suggested high atherogenicity of Lp(a), its susceptibility for Cu(II)-induced oxidation was studied and compared with that of LDL. Since the content of antioxidants as well as the fatty acid pattern of a lipoprotein greatly affects its oxidizability, Lp(a) and LDL were characterized first with respect to these substances. Paired samples of low-density lipoproteins (LDL) and Lp(a) were isolated from seven individual donors and compared with each other. This study showed that LDL and Lp(a) are very similar with respect to their fatty acid and antioxidant composition. LDL contains approx. 1132 nmol of total fatty acids/mg lipoprotein and LDL 1466 nmol total fatty acids/mg lipoprotein. Analysis of the fatty acid composition of individual lipid classes (cholesteryl esters, phospholipids and triacylglycerols) revealed also a high similarity in the composition of these lipid classes between the two lipoproteins. A comparison of the antioxidant composition showed that Lp(a) contains less alpha-tocopherol than LDL (1.6 +/- 0.35 nmol/mg vs. 2.1 +/- 0.25 nmol/mg LDL). In copper(II)-induced lipid peroxidation experiments we found a striking difference in the susceptibility of individual lipoprotein classes between all donors. In addition, Lp(a) exhibited a 1.2 to 2.4 longer lag-phase than the corresponding LDL preparation from the same blood donor. Treatment of Lp(a) with neuraminidase resulted in a drastic decrease of the lag-phase of Lp(a). Neuraminidase treatment of LDL on the other hand had no significant effects on its susceptibility to oxidation. Supplementation of neuraminidase-treated Lp(a) with N-acetylneuraminic acid (NANA) at concentrations comparable to the naturally occurring amounts of NANA in the Lp(a) protein moiety led to an increase of the lag-phase yielding values which were comparable to those observed with native Lp(a). These results demonstrate that the fatty acid composition as well as the antioxidant concentrations of Lp(a) and LDL are quite similar; despite this fact, Cu2(+)-mediated oxidation of Lp(a) is retarded in comparison to LDL which might be due to the higher content of NANA in Lp(a).

Adult↗
PubMed1991

Determination of fatty acids in the main lipoprotein classes by capillary gas chromatography: BF3/methanol transesterification of lyophilized samples instead of Folch extraction gives higher yields.

The amount of individual fatty acids contained in the main human lipoproteins VLDL, LDL, lipoprotein (a), HDL2, and HDL3 were determined by two different methods. In Method I, the lipids were first extracted by the classical Folch procedure and then transesterified with BF3/methanol and separated by capillary GC. In Method II the lipoprotein solution was freeze dried prior to transesterification with BF3/methanol. In all lipoproteins except VLDL significantly more fatty acids were found with Method II as compared to Method I. For total fatty acids the increase was up to 17.5%, for polyunsaturated fatty acids up to 24.5%. The total fatty acid content determined by Method II resembled closely the content independently derived from the enzymatically determined lipid composition. The results indicate that in case of lipoproteins quantification of fatty acids should be made with freeze-dried samples rather than with Folch extracts.

Blood Proteins↗
PubMed1991

Platelet membrane fluidity in type IIA, type IIB and type IV hyperlipoproteinemia.

Fluorescence spectroscopy, a very sensitive index for measuring the biophysical properties of living cell systems, was used to examine the structural order of intact, resting, gel-filtered platelets from hyperlipidemic subjects (n = 48, 25-70 years) and normolipemic subjects (n = 34, 19-68 years). Fluorescence anisotropy (r[s]), which is inversely related to membrane fluidity, was estimated using 3 different fluorescent dyes, DPH, TMA-DPH, and 6-AS, known to label different regions of biological membranes. Increased membrane fluidity was observed in type IIB (n = 24, 36-62 yrs; r[s] = 0.0692 +/- 0.09) and type IV (n = 10, 33-57 yrs; r[s] = 0.058 +/- 0.006) hyperlipidemics in comparison to type IIA (n = 14, 25-70 yrs; r[s] = 0.086 +/- 0.019) and control subjects (n = 24, 28-68 yrs; r[s] = 0.079 +/- 0.012). The temperature dependency of r[s]-DPH values was significantly different (P less than 0.01) in platelets from type IIB and type IV patients compared to type IIA and control subjects of similar age. A significant positive correlation (P less than 0.005) between membrane fluidity and age was found only in healthy control subjects (n = 34, 19-68 yrs). Despite significant (P less than 0.01) differences in plasma lipid concentrations in hyperlipidemic patients and controls, significant ex vivo relations between membrane fluidity and lipoprotein concentrations, free fatty acid distribution, and increased age were found only in healthy control subjects. Plasma levels of thromboxane as well as serum selenium concentrations did not significantly differ between hypercholesterolemic, hypertriglyceridemic, and control subjects.

Adult↗
PubMed1991

Effects of dietary fish oil supplementation on platelet aggregability and platelet membrane fluidity in normolipemic subjects with and without high plasma Lp(a) concentrations.

The purpose of this study was to compare the relative effect of n-3 fatty acids on plasma lipids and platelet function in normolipemic subjects (n = 8) with plasma Lp(a) levels greater than 30 mg/dl and normolipemic subjects (n = 7) without detectable plasma Lp(a) concentrations. Six weeks of dietary supplementation (3.8 g EPA and 2.9 g DHA/d) significantly reduced (P less than 0.005) plasma TGs in both groups whereas no changes of plasma TC, LDL-C, HDL-C, and Lp(a), respectively, were found. Collagen- or thrombin-stimulated platelet aggregation and collagen- or thrombin-induced TXB2 generation from platelets decreased by approx. 45% in Lp(a)-negative and Lp(a)-positive platelet donors after a 6 week dietary intake. Four more weeks without n-3 supplementation restored the pretreatment values of TGs, platelet aggregability and TXB2 release. The biophysical properties of platelets from normolipemics with and without high plasma Lp(a) concentrations revealed a similar structural order of platelets at 37 degrees C using DPH, TMA-DPH, or 6-AS as fluorescent probes. Also similar temperature-dependent changes in platelet fluidity from 37 degrees C to 17 degrees C were observed in platelet preparations from Lp(a)-positive and Lp(a)-negative subjects. However, no subtle changes in the structural order of platelets due to nutrient intakes were found in all subjects (n = 15, 19-28 yrs) using fluorescence polarization technique. The present data suggest a similar in vitro platelet behaviour from normolipemic subjects with and without high plasma levels of Lp(a) (which is considered a risk for premature atherosclerosis) in contrast to platelet aggregability and platelet fluidity in certain hyperlipidemic stages.

Adult↗
PubMed1991

Oxidized low-density lipoprotein antagonizes the activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor but does not interfere with its biosynthesis.

Oxidized low-density lipoprotein (LDLox) is a molecule with strong atherogenic properties. In a concentration dependent fashion, LDLox antagonized the activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor (EDRF), which was produced in vitro by incubation of a partially purified EDRF-forming enzyme in the presence of L-arginine, Ca2+ and NADPH. The inhibitory effect of LDLox was potentiated by preincubation of the soluble guanylate cyclase with LDLox, but not when the EDRF-forming enzyme was pretreated with LDLox. As LDLox did not diminish the calmodulin-dependent conversion of L-arginine into L-citrulline by the EDRF-forming enzyme it would appear that EDRF-biosynthesis was not affected by LDLox. It is suggested that the impaired relaxant response of atherosclerotic blood vessels to endothelium-dependent vasodilators was not due to a reduced formation of EDRF but due to a diminished responsiveness of soluble guanylate cyclase.

Animals↗