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G M Kostner

Publications and source records attributed to G M Kostner.

At least 73 records · Page 4Linked to original sources
PubMed1997

Activation of microsomal cytochrome P450 mono-oxygenase by Ca2+ store depletion and its contribution to Ca2+ entry in porcine aortic endothelial cells.

1. We investigated how microsomal cytochrome P450 mono-oxygenase (Cyp450 MO) is regulated in cultured porcine aortic endothelial cells. The hypothesis that a Cyp450 MO-derived metabolite links Ca2+ store depletion and Ca2+ entry was studied further. 2. Microsomal Cyp450 MO was monitored fluorometrically by dealkylation of 1-ethoxypyrene-3,6,8-tris-(dimethyl-sulphonamide; EPSA) in saponin permeabilized cells or in subcellular compartments. Endothelial Ca2+ signalling was measured by a standard fura-2 technique, membrane potential was determined with the potential-sensitive fluorescence dye, bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(5)) and tyrosine kinase was quantified by measuring the phosphorylation of a immobilized substrate with a horseradish peroxidase labelled phosphotyrosine specific antibody. 3. Depletion of cellular Ca2+ pools with inositol 1,4,5-trisphosphate (IP3), thapsigargin or cyclopiazonic acid activated microsomal Cyp450 MO. Similar to direct Ca2+ store depletion, chelating of intramicrosomal Ca2+ with oxalate stimulated Cyp450 MO activity, while changing cytosolic free Ca2+ failed to influence Cyp450 MO activity. These data indicate that microsomal Cyp450 MO is activated by depletion of IP3-sensitive stores. 4. Besides the common cytochrome P450 inhibitors, econazole, proadifen and miconazole, thiopentone sodium and methohexitone inhibited Cyp450 MO in a concentration-dependent manner. The physiological substrate of Cyp450 MO, arachidonic acid, inhibited EPSA dealkylation. In contrast to most other cytochrome P450 inhibitors used in this study, thiopentone sodium did not directly interfere with Ca2+ entry pathways, membrane hyperpolarization due to K+ channel activation or tyrosine kinase activity. 5. Inhibition of Cyp450 MO by thiopentone sodium diminished Ca2+/Mn2+ entry to Ca2+ store depletion by 43%, while it did not interfere with intracellular Ca2+ release by IP3 or thapsigargin. 6. Cyp450 MO inhibition with thiopentone sodium diminished autacoid-induced membrane hyperpolarization. 7. Induction of Cyp450 MO with dexamethasone/clofibrate for 72 h yielded increases in thapsigargin-induced Cyp450 MO activity (by 35%), Ca2+/Mn2+ entry (by 105%) and membrane hyperpolarization (by 40%). 8. The Cyp450 MO-derived compounds, 11,12 and 5,6-epoxyeicosatrienoic acids (EETs) yielded membrane hyperpolarization, insensitive to thiopentone sodium. 9. These data demonstrate that endothelial Cyp450 MO is activated by Ca2+ store depletion and Cyp450 MO produced compounds that hyperpolarize endothelial cells. 10. The data presented and our previous findings indicate that Cyp450 MO plays a crucial role in the regulation of store-operated Ca2+ influx. We propose that Cyp450 MO-derived EETs constitute a signal for Ca2+ entry activation and increase the driving force for Ca2+ entry by membrane hyperpolarization in porcine aortic endothelial cells.

8,11,14-Eicosatrienoic Acid↗
PubMed1997

Role of superoxide anions in changes of endothelial vasoactive response during acute hyperglycemia.

The effects of acute hyperglycemia on endothelial Ca2+ signaling, formation of endothelium-derived relaxing factor (EDRF) and bioactivity of EDRF were investigated. Hyperglycemia increased 2,5-tert-butyl-1,4-hydrochinone (BHQ)-initiated Ca2+ signaling and EDRF formation in a concentration-dependent manner. The effect of elevated D-glucose on Ca2+/EDRF response could be diminished by co-incubation with the antioxidants vitamin E, probucol, GSH, vitamin C and superoxide dismutase. Convincingly, hyperglycemic conditions yielded an increase in superoxide anion release from endothelial cells and the superoxide anion-generating mixture xanthine oxidase/hypoxanthine mimicked the effect of hyperglycemia on Ca2+/EDRF signaling. Besides an enhanced formation of the vasodilatatory NO compound EDRF, hyperglycemia enhanced NO degradation by endothelial cells and, thus, reduced bioactivity of EDRF. We suggest that vasoactivity during acute hyperglycemia depends on the superoxide anion scavenging properties of the vascular wall. In acute hyperglycemia and early stages of diabetes, radical scavenging capacity may be suitable to protect NO degradation, resulting in an enhanced vasodilation. In contrast, decreased free radical scavenging properties of the vasculature in prolonged hyperglycemia and in later stages of diabetes might promote NO degradation by an overshoot of superoxide anions, resulting in an attenuation of endothelium-dependent vasodilation.

Animals↗
PubMed1997

The role of apo-(a) kringle-IVs in the assembly of lipoprotein-(a).

Lipoprotein-(a) [Lp(a)] is a highly atherogenic lipoprotein with unknown function, consisting of a low-density lipoprotein (LDL) core and the apo(a) glycoprotein. The characteristic structural feature of apo(a) is the presence of multiple so called "kringle' repeats which are in part identical and in part exhibit slight sequence differences. The assembly of apo(a) and LDL, which is determinant for plasma Lp(a) levels, takes place extracellularly and requires specific structural motifs in apo(a) and apoB. Here we studied the structural features in apo(a) necessary for high-efficient assembly. Thirteen recombinant apo(a) glycoproteins, which differed in the set of kringle-IV (K-IV) motifs, were expressed in COS-7 cells and incubated with LDL. The rate of total and disulfide-stabilized Lp(a) complex formation was measured by an immunochemical assay. Constructs containing K-IV T(type)5-T10 yielded almost 100% total and 80% stable complexes, respectively. Deletion or replacement of the different kringles revealed that K-IV T6 and T7 were responsible for the high-yield assembly and that K-IV T5 had an amplifying effect. Increasing the absolute number of K-IV repeats had an additional amplifying effect. The rate of Lp(a) assembly correlated strongly with the affinity of these constructs to Lys-Sepharose. Our results have implications for understanding the metabolism of Lp(a) and may help to design strategies for searching natural apo(a) mutants with aberrant plasma Lp(a) levels.

Animals↗
PubMed1997

Metabolism of Lp(a): assembly and excretion.

Lp(a) is one of the most atherogenic lipoproteins, and we know much more about the pathophysiology of Lp(a) than about its physiological function and metabolism. From our previous investigations and the new results reported here, we propose the following model of Lp(a) metabolism: apo(a) is biosynthesized in liver cells and the size of the isoform determines its rate of synthesis and excretion. Specific kringle-4 domains in apo(a), mainly T-6 and T-7, bind in a first step to circulating LDL, followed by the stabilization of the newly formed Lp(a) complex by a disulfide bridge. Circulating Lp(a) interacts specifically with kidney cells, or possibly other tissues, causing cleavage of 2/3-3/4 of the N-terminal part of apo(a) by a collagenase-type protease. Part of the apo(a) fragments is found in the urine, but there are indications that they in fact represent the biologically active form of apo(a). The core portion of Lp(a) in turn is cleared by the LDL-receptor or another specific binding system of the liver. Strategies for reducing plasma Lp(a) levels with medication should aim at interfering with the assembly of Lp(a) on one hand and the stimulation of apo(a) fragmentation on the other hand.

Animals↗
PubMed1997

Standardization of Lp(a) measurements.

The standardization of immunoassays for Lp(a) is a major challenge to clinical chemists. In order to establish a reference material acknowledged by the International Federation of Clinical Chemistry, the Center of Disease Control and possibly the World Health Organization, a working group with participants from four continents was put together. With the aid of 34 companies, eight proposed reference materials have been tested in the last 3 years and two of them have been selected for value assignment. A reference method based on dissociation-enhanced lanthanide fluorescence immunoassays was therefore developed which gives linear and parallel response curves by assaying freshly prepared primary reference standards, fresh plasma or serum as well as lyophilized or frozen proposed reference material. For value assignment, four laboratories simultaneously prepare primary reference standards with known isoforms and molecular weights. By assaying the amino acid composition of these primary reference standards, the molar concentration which is the basis of value transfer to the lyophilized proposed reference material can be calculated. In a final step, harmonization of all commercially available Lp(a) kits is to be tested by assaying 50 Lp(a) samples with increasing Lp(a) concentrations and varying isoforms. We hope to be able in the near future to create a basis for comparable results in epidemiological studies in different laboratories and also to help to improve future long-term precision in clinical chemical laboratories.

Centers for Disease Control and Prevention, U.S.↗
PubMed1997

Apolipoprotein E polymorphism and silent microangiopathy-related cerebral damage. Results of the Austrian Stroke Prevention Study.

BACKGROUND AND PURPOSE: Microangiopathy-related cerebral damage (MARCD) includes white matter abnormalities and lacunar infarctions and represents a common MRI observation in subjects above 50 years of age. The risk factors of such brain abnormalities are not fully determined. The goal of this study was to determine whether the genetic heterogeneity of apolipoprotein E (apoE) contributes to the occurrence of MARCD. METHODS: Brain MRI (1.5 T) was performed in 280 individuals (ages 50 to 75 years) without neuropsychiatric disease randomly selected from the official register of residents of the city of Graz, Austria. All study participants underwent apoE genotyping, carotid Doppler sonography, electrocardiography, echocardiography, and a complete blood chemistry panel. MARCD was defined as evidence of early confluent and confluent white matter hyperintensities or lacunes. Carotid atherosclerosis was graded on a five-point scale ranging from not present (0) to complete occlusion (5). RESULTS: MARCD occurred in 61 individuals (21%). The distribution of apoE genotypes differed significantly between subjects with and without MARCD (P = .036). Subjects with such findings more commonly had the epsilon 2/epsilon 3 genotype (24.6% versus 10%) at similar frequencies of genotypes containing the epsilon 4 allele. The epsilon 2/epsilon 3 genotype was associated with lower levels of total cholesterol (P = .0009), LDL cholesterol (P = .00001), and apolipoprotein B (P = .00001). Also, there was a nonsignificant trend toward less cardiac disease. Other major vascular risk factors and carotid abnormalities were similar among the various genotypes. Multiple logistic regression analysis created a model of significant MARCD predictors, including age (odds ratio [OR], 1.1 per year), hypertension (OR, 3.4), and the apoE epsilon 2/epsilon 3 genotype (OR, 3.0). CONCLUSIONS: These data suggest an association between the apoE epsilon 2/epsilon 3 genotype and MARCD despite favorable effects on the lipid profile and cardiac disease.

Aged↗
PubMed1997

[Prevalence and risk factors in the population of Graz (Austrian Stroke Prevention Study)].

The Austrian Stroke Prevention Study recruited 1960 randomly selected subjects aged 50 to 75 years during a 3-year period of enrollment. The response rate of the study was 32.4%. A telephone interview with 200 randomly selected non-responders yielded no differences to responders regarding the frequency of major vascular risk factors known to the subjects. Besides demographics, the study assessed arterial hypertension, diabetes mellitus, cardiac disease, smoking, a complete lipid status including the apolipoprotein-E genotype, serum fibrinogen and anticardiolipin antibodies as well as various natural antioxidants such as vitamins A, C, E and beta-carotene. Arterial hypertension, diabetes mellitus, cardiac disease and hypercholesterolemia > 200 mg/dl were strikingly common and occurred in 38%, 7.6%, 32% and 76%, respectively. Suboptimal plasma concentrations of vitamin A, E, and beta-carotene were noted in 77.2%, 56.1% and in 53.2% of study participants. The rate of treatment of major risk factors known to the subjects prior to study entry were 60.3% and 70% for arterial hypertension and diabetes mellitus, but only 37.1% and 6.3% for cardiac disease and hypercholesterolemia > 250 mg/dl. Diet was commonly used to treat diabetes but was almost neglected in the treatment of other vascular risk factors. These data provide an orientation on the prevalence of risk factors and the use of primary preventive measures for stroke treatment in our community.

Adult↗
PubMed1997

Implications of endogenous and exogenous lipoprotein lipase for the selective uptake of HDL3-associated cholesteryl esters by mouse peritoneal macrophages.

To investigate the implications of endogenous LPL on selective uptake of HDL3-associated cholesteryl esters (HDL3-CEs) by mouse peritoneal macrophages (MPMs), we have performed uptake experiments with MPMs obtained from control mice and transgenic knockout animals expressing LPL exclusively in muscle but not in macrophages. The capacity for HDL3 holoparticle, total HDL3-CE, and selective HDL3-CEs was independent of the expression of functional endogenous LPL (161 vs. 187, 1251 vs. 1300, and 1900 vs. 1113 ng HDL3/mg cell protein; control and LPL-deficient macrophages, respectively). Both control and LPL-deficient macrophages displayed, however, pronounced capacity for total HDL3-CE uptake in excess of HDL3 holoparticle uptake exceeding particle uptake by 7-fold. Despite the fact that endogenous LPL was without any effect on selective uptake, the addition of exogenous LPL led to a significant increase in cellular selective HDL3-CE uptake. Upon addition of purified LPL, HDL3 holoparticle (internalization and degradation), total HDL3-CE, and selective HDL3-CEs, was increased up to 2-fold. HDL3 holoparticle binding to control and LPL-deficient MPMs at 4 degrees C was enhanced 2.7- and 2.6-fold, respectively, in the presence of LPL. The present results suggest that endogenous LPL is without effect on selective uptake of HDL3-CEs. In contrast, the addition of exogenous LPL enhanced selective uptake of HDL3-CEs along with HDL3 holoparticle uptake apparently by the proposed bridging function of the enzyme.

Animals↗
PubMed1997

LDL-mediated interaction of Lp[a] with HepG2 cells: a novel fluorescence microscopy approach.

We studied the topography of Lp[a]-LDL-cell interactions by means of fluorescence microscopy, using fluorescence-labeled lipoproteins. In contrast to known methods which are based on noncovalent labeling of lipoproteins by positively charged amphiphiles, the protein moiety of LDL and Lp[a] was covalently labeled with either BODIP-succinimide-ester (green) or rhodamine X iodoacetamide (red). The interaction of the fluorescent lipoproteins with cultured HepG2 cells was studied using a confocal laser scanning fluorescence microscope. LDL and Lp[a], each labeled with a different dye, could be examined separately within a mixture of both lipoproteins during their interaction with HepG2 cells. At 4 degrees C, the majority of both fluorescent particles co-localized and only a few separate LDL- or Lp[a]-binding domains could be observed. Quantification of the amount of fluorescent lipoprotein associated with the cell surface at 4 degrees C showed that binding of Lp[a] was increased in the presence of LDL under these conditions, probably via formation of an Lp[a]-LDL complex. At 37 degrees C, LDL and Lp[a] were taken up by the cells within 10 min. Again the majority of LDL and Lp[a] particles co-localized intracellularly. Only minor amounts of LDL and Lp[a] could be observed separately. As the entire fluorescence of labeled Lp[a] co-localized with excess of LDL in cells, and taking into account the high tendency of LDL-Lp[a] association in solution and on cell surfaces, it is concluded that a significant portion of the internalized Lp[a] is taken up into the cells by the LDL receptor via LDL by a hitchhiking-like process.

Animals↗
PubMed1996

Increased selective uptake in vivo and in vitro of oxidized cholesteryl esters from high-density lipoprotein by rat liver parenchymal cells.

Oxidation of low-density lipoprotein (LDL) leads initially to the formation of LDL-associated cholesteryl ester hydroperoxides (CEOOH). LDL-associated CEOOH can be transferred to high-density lipoprotein (HDL), and HDL-associated CEOOH are rapidly reduced to the corresponding hydroxides (CEOH) by an intrinsic peroxidase-like activity. We have now performed in vivo experiments to quantify the clearance rates and to identify the uptake sites of HDL-associated [3H]Ch18:2-OH in rats. Upon injection into rats, HDL-associated [3H]Ch18:2-OH is removed more rapidly from the circulation than HDL-associated [3H]Ch18:2. Two minutes after administration of [3H]Ch18:2-OH-HDL, 19.6 +/- 2.6% (S.E.M.; n = 4) of the label was taken up by the liver as compared with 2.4 +/- 0.25% (S.E.M.; n = 4) for [3H]Ch18:2-HDL. Organ distribution studies indicated that only the liver and adrenals exhibited preferential uptake of [3H]Ch18:2-OH as compared with [3H]Ch18:2, with the liver as the major site of uptake. A cell-separation procedure, employed 10 min after injection of [3H]Ch18:2-OH-HDL or [3H]Ch18:2-HDL, demonstrated that within the liver only parenchymal cells take up HDL-CE by the selective uptake pathway. Selective uptake by parenchymal cells of [3H]Ch18:2-OH was 3-fold higher than that of [3H]Ch18:2, while Kupffer and endothelial cell uptake of the lipid tracers reflected HDL holoparticle uptake (as analysed with iodinated versus cholesteryl ester-labelled HDL). The efficient uptake of [3H]Ch18:2-OH by parenchymal cells was coupled to a 3-fold increase in rate of radioactive bile acid secretion from [3H]Ch18:2-OH-HDL as compared with [3H]Ch18:2-HDL. In vitro studies with freshly isolated parenchymal cells showed that the association of [3H]Ch18:2-OH-HDL at 37 degrees C exceeded [3H]Ch18:2-HDL uptake almost 4-fold. Our results indicate that HDL-associated CEOH are efficiently and selectively removed from the blood circulation by the liver in vivo. The selective liver uptake is specifically exerted by parenchymal cells and coupled to a rapid biliary secretion pathway. The liver uptake and biliary secretion route may allow HDL to function as an efficient protection system against potentially atherogenic CEOOH.

Animals↗
PubMed1996

The relative electrophoretic mobility of apo(a) isoforms depends on the gel system: proposal of a nomenclature for apo(a) phenotypes.

Genetic apo(a) isoforms were originally defined according to their relative mobility in SDS-PAGE compared to apoB-100 and were designated as F, B or S1-S4 isotypes. This widely accepted nomenclature does not accommodate the broad spectrum of apo(a) isoforms (> 30) detected by high resolution SDS-agarose gel electrophoresis. Moreover we here show that the relative mobilities of apo(a) isoforms depend on the SDS-gel system used. Comparison of the SDS-PAGE system originally used for phenotyping with SDS-agarose gel electrophoresis and two commercial SDS-PAGE systems (PhastGel, Pharmacia, Sweden and NOVEX, USA) demonstrated marked differences in resolving power and resulted in very different Rf values for identical isoforms. Hence phenotyping results from laboratories using different systems are not comparable. We therefore propose a nomenclature of apo(a) isoforms which reports the number of kringle IV repeats in the apo(a) allele (e.g. apo(a) K-IV20 would designate an isoform with 20 K-IV repeats). This is achieved by using standards in which the number of kringle IV repeats has been determined by pulsed field gel electrophoresis of genomic DNA. The proposed nomenclature (i) accounts for the increased resolution of apo(a) phenotyping methods: (ii) is flexible to the introduction of smaller or larger isoforms; (iii) allows to report data from systems with lower resolution as 'binned' isoform categories; (iv) allows the comparison of phenotyping results between different investigators; and (v) can be applied on DNA as well as on protein based apo(a) phenotyping.

Apolipoproteins A↗
PubMed1996

Muscle-specific overexpression of lipoprotein lipase in transgenic mice results in increased alpha-tocopherol levels in skeletal muscle.

Lipoprotein lipase (LPL) has been implicated in the delivery of chylomicron-located alpha-tocopherol (alpha-TocH) to peripheral tissues. To investigate the role of LPL in the cellular uptake of alpha-TocH in peripheral tissue in vivo, three lines of transgenic mice [mouse creatine kinase- (MCK) L, MCK-M and MCK-H] expressing various amounts of human LPL were compared with regard to alpha-TocH levels in plasma, skeletal muscle, cardiac muscle, adipose tissue and brain. Depending on the copy number of the transgene, LPL activity was increased 3- to 27-fold in skeletal muscle and 1.3- to 3.7-fold in cardiac muscle. The intracellular levels of alpha-TocH in skeletal muscle were significantly increased in MCK-M and MCK-H animals and correlated highly with the tissue-specific LPL activity (r = 0.998). The highest levels were observed in MCK-H (21.4 nmol/g) followed by MCK-M (13.3 nmol/g) and MCK-L (8.2 nmol/g) animals when compared with control mice (7.3 nmol/g). Excellent correlation was also observed between intracellular alpha-TocH and non-esterified fatty acid (NEFA) levels (r = 0.998). Although LPL activities in cardiac muscle were also increased in the transgenic mouse lines, alpha-TocH concentrations in the heart remained unchanged. Similarly, alpha-TocH levels in plasma, adipose tissue and brain were unaffected by the tissue specific over-expression of LPL in muscle. The transgenic model presented in this report provides evidence that the uptake of alpha-TocH in muscle is directly dependent on the level of LPL expression in vivo. Increased intracellular alpha-TocH concentrations with increased triglyceride lipolysis and NEFA uptake might protect the myocyte from oxidative damage during increased beta-oxidation.

Adipose Tissue↗
PubMed1996

Cholesterol metabolism in cells with different peroxisomal defects.

We showed previously that cholesterol biosynthesis in dermal fibroblasts from patients with metabolic disorders of peroxisomal origin is increased in steps prior to mevalonate, whereas low-density-lipoprotein(LDL)-receptor activities were not different from control fibroblasts. Here, the suppression of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity by lovastatin was studied both in dermal fibroblasts from patients with different peroxisomal defects and in a Chinese hamster ovary (CHO) cell line lacking morphologically intact peroxisomes. In addition, the formation of intracellular cholesteryl esters (a measure of acyl-CoA:cholesterol acyltransferase(ACAT)-activity) stimulated by exogenous LDL was investigated. A dose-dependent suppression of cholesterol biosynthesis by lovastatin at concentrations of 1-50 mumol/l was observed which was comparable in normal and peroxisomal-disease fibroblasts. ACAT activity was measured in the absence and presence of exogenous LDL using [3H]oleate as a substrate for cholesterol ester synthesis. The basal esterification rate was equal or lower in peroxisomal-defective fibroblasts compared with controls. In the presence of exogenous LDL, cholesterol esterification was significantly impaired in all defective cells in comparison with normal fibroblasts. We conclude that changes in cholesterol homeostasis in peroxisomal diseased fibroblasts be related to cholesterol ester formation.

Animals↗
PubMed1996

Alpha-tocopherol and hydroperoxide content in breast adipose tissue from patients with breast tumors.

The study of the relationship between dietary intake of vitamin E and the risk of breast cancer has not yielded definite conclusions with respect to causality, possibly due to methodological issues inherent to nutritional epidemiology. To avoid the pitfalls of dietary recalls, alpha-tocopherol content of adipose tissue was used as a biochemical indicator of long-term dietary intake of vitamin E. alpha-tocopherol and hydroperoxides were measured in breast adipose tissue obtained at the time of diagnosis from 70 patients with early breast cancer. Thirty women with non-malignant breast tumors served as control. Lipid peroxidation was monitored by quantifying conjugated dienes spectrophotometrically and by assaying hydroperoxides with an iodometric method; alpha-tocopherol was measured by HPLC associated with fluorescence detection. Mean alpha-tocopherol value in breast adipose tissue was significantly lower in breast cancer patients than in control patients, whereas the hydroperoxide content was significantly higher in cancer patients than in controls. The alpha-tocopherol concentration in adipose tissue was not correlated with the clinical status of the patients with respect to age, menopausal status or body mass index. We conclude from this pilot study that breast cancer is associated with a low content of alpha-tocopherol in breast adipose tissue, and with an altered lipid oxidation pattern, which might be related to a low antioxidant status.

Adipose Tissue↗
PubMed1996

Quantification of lipoprotein lipase (LPL) by dissociation-enhanced lanthanide fluorescence immunoassay. Comparison of immunoreactivity of LPL mass and enzyme activity of LPL.

Lipoprotein lipase (LPL) hydrolyses triglycerides in chylomicrons and in very low density lipoproteins. In this study, a new sensitive enzyme immunoassay, the dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), for the quantification of immunoreactive LPL mass in biological specimens was developed. In the indirect sandwich DELFIA assay polyclonal anti-human or anti-bovine LPL IgGs were used as capture antibodies, monoclonal antibody (mAb) 5D2 and Eu(3+)-labelled goat anti-mouse IgG were used as detection antibodies. In the direct sandwich DELFIA assay, mAb 5D2 was used as capture and Eu(3+)-labelled mAb 5D2 as detection antibodies. Both purified bovine and human LPL proteins served as standards in the indirect and the direct DELFIA assay. Standard curves were linear between 0.1 and 1000 ng LPL/ml, assuring the sensitivity of the DELFIAs within this range. Mean values for immunoreactive LPL mass in normal individuals were found to be 40.3 +/- 14.4 ng/ml preheparin plasma and 334.1 +/- 71 ng/ml postheparin plasma. In patients affected with type I hyperlipoproteinemia 82.4 +/- 29.3 ng/ml (postheparin plasma) were determined. Coefficients of inter- and intra-assay variation were 4.3% and 6.2% on average. The correlation coefficient between the indirect and the direct DELFIA technique was 0.9694. The correlation coefficient between immunoreactive LPL mass (estimated by DELFIA) and LPL activity (estimated by the LPL activity assay) was 0.9345. Our data are consistent with the concept that LPL is active as a dimer. Dissociation of the LPL dimer into monomers is tightly coupled to both loss of immunoreactivity and enzyme activity of LPL.

Animals↗
PubMed1996

Photometric evaluation of lipid peroxidation products in human plasma and copper oxidized low density lipoproteins: correlation of different oxidation parameters.

Despite considerable methodological advances, ideal parameters relating to the in vitro assessment of lipoprotein oxidizability are lacking. In this study, some of the more common parameters of lipid peroxidation were measured in 30 plasma samples. The following parameters were determined: conjugated dienes (method 1), reaction of lipid hydroperoxides with a methylene blue derivative (method 2), oxidation of iodide to triiodide (method 3) and an iodometric assay based on the same chemistry but modified to correct for unspecific interferences (method 4). Alpha-Tocopherol in plasma was assayed by high-pressure liquid chromatography. In addition, LDL was isolated from plasma and the susceptibility of individual LDL preparations towards copper-initiated oxidation was characterized. The amount of lipid hydroperoxides found in fresh plasma samples obtained from apparently healthy humans was dependent on the method used for the assessment. Lipid hydroperoxides measured by method 2 were: 8.6 +/- 5.8 mu M, by method 3: 5.8 +/- 1.9 mu M and by method 4: 4.2 +/- 2.7 mu M. Mean values of conjugated dienes (method 1) were 84.6 +/- 20.9 mu M; The content of alpha-tocopherol in plasma was 23.6 +/- 3.9 mu M. Despite the differences in absolute values, a statistically significant correlation was found between values obtained by methods 1, 2 and 3, but not by method 4. An inverse relationship has been observed between the lipid hydroperoxide content in plasma obtained with method 4 and two parameters of LDL oxidation (diene concentration, rate of diene formation) but not with the lag time. Our data suggest that--among the photometric methods evaluated--method 4 might be the most specific for the measurement of plasma (and lipoprotein associated) lipid hydroperoxides.

Adult↗
PubMed1996

Preparation of fatty acid methyl esters from lipoprotein and macrophage lipid subclasses on thin-layer plates.

A simple, accurate, and fast procedure for quantitative analysis of fatty acids (FA) in simple lipid subclasses from different biological specimens is presented. Lipid extracts of isolated plasma lipoproteins (very low, low, and high density lipoproteins; VLDL, LDL, and HDL, respectively) and permanent J774 mouse macrophages were fractionated into lipid subclasses by thin-layer chromatography (TLC) on silica gel 60 plates. Bands comigrating with authentic lipid standards were scraped off under argon and subjected to direct, in situ transesterification with BF3/MeOH in the presence of the TLC adsorbent. Fatty acid methyl esters were subsequently quantitated by capillary gas chromatography. A comparison of the FA content present in total lipid extracts and in lipid subclasses separated by TLC revealed recoveries ranging from 93 (J774 cell extracts) to 99.7% (LDL). The method described is applicable for the measurement of FA in individual lipid subclasses and was successfully applied to quantitatively analyze the FA composition of the phospholipid, triacylglycerol, and cholesteryl ester fraction derived from VLDL, LDL, and HDL. In J774 lipid extracts, the FA composition of the phospholipid-, monoacylglycerol-, diacylglycerol-, free fatty acid-, triacylglycerol-, and cholesteryl ester fraction was quantitated. In addition we have analyzed the time-dependent loss of the major HDL polyunsaturated fatty acids (18:2, 20:4) in the phospholipid and cholesteryl ester fraction during copper-dependent peroxidation of HDL. We have not encountered analytical problems concerning low FA recoveries from CE-rich lipid extracts as indicated by almost quantitative recoveries of FA in LDL, HDL, and J774 extracts.

Animals↗