Physical,chemical and immunological characteristics of continuous-density lipoproteins isolated by single-spin gradient ultracentrifugation

The isolation of various lipoproteins of very low, intermediate, low and high density from 5 ml of serum was obtained by single-step ultracentrifugation in a potassium bromide density gradient obtained by smoothing a discontinuous gradient during the run (120 000g, 48 h, 4°C; Beckman SW27 rotor). The density profiles were reproducible. The lipoproteins were characterized by electron microscopy, fluorescence polarization, polyacrylamide gel electrophoresis and complete chemical assays. The composition of the entire lipoprotein spectrum was in accordance with previous results. The lipoprotein molecular mass, particle size distribution, Apo AI/AII ratio and microviscosity varied according to the density range.     

Anion-exchange HPLC separation of five major rabbit lipoproteins using a nonporous diethylaminoethyl-ligated gel with a perchlorate-containing eluent

Rabbits are often used as experimental animals in studies of atherosclerosis and hyperlipidemia. Although rabbit lipoproteins can be quantitated by sequential ultracentrifugation, a simpler and more reproducible method is desirable for detailed analyses. The current study describes a method to analyze rabbit lipoproteins in plasma by anion‐exchange high‐performance liquid chromatography using a column filled with nonporous, diethylaminoethyl‐ligated polymers. A solution of NaClO₄ was used to adjust the ionic strength of the eluent. The method required only 15 μL of plasma and analysis was completed in 23 min. Five lipoprotein fractions (high‐density lipoprotein, low‐density lipoprotein, intermediate‐density lipoprotein, very‐low‐density lipoprotein and chylomicrons) were eluted with step‐wise increases in a concentration of NaClO₄. The post‐column eluate was reacted with an enzymatic reagent to determine total cholesterol, and the lipoprotein‐cholesterol fraction was calculated according to relative peak areas in the chromatogram. The within‐day and between‐day assay coefficients of variation for lipoprotein cholesterol levels ranged between 0.436 and 7.143% and between 2.905 and 10.526%, respectively. Administering a high‐fat diet increased lipoprotein‐cholesterol concentrations by 6‐ to 77‐fold. The method described here was nevertheless able to quantitate levels of lipoprotein‐cholesterol in plasma samples from these rabbits. These results indicate that this method may be applied to lipoprotein studies using hyperlipidemic rabbit models. Copyright © 2011 John Wiley & Sons, Ltd.     

Purification of serum amyloid A and its isoforms from human plasma by hydrophobic interaction chromatography and preparative isoelectric focusing.

The present work was aimed at isolating human serum amyloid A, (SAA), an acute-phase protein mainly complexed to high density lipoproteins, directly from human plasma without sequential ultracentrifugation of lipoproteins and subsequent delipidation of the apolipoprotein moiety. Hydrophobic-interaction fast-protein liquid chromatography on Octylsepharose, using stepwise gradient elution profiles under dissociating conditions, followed by fast-protein liquid-gel permeation chromatography on a Superdex TM75 column revealed a higher than 95% purity of isolated SAA. Further purification of SAA from coeluting apolipoproteins C and A-II was achieved by preparative isoelectric focusing between pH5-7 using a Rotofor apparatus. Separation of the main SAA isoforms, SAA1 (pI 6.5) and SAA1 des-Arg (pI 6.0, lacking the N-terminal arginine), was achieved by anion-exchange fast-protein liquid chromatography on a Fractogel EMD DEAE 650-S column. The purity of the SAA1 and SAA1 des-Arg isoforms, thus isolated, was checked by immunochemical techniques and amino acid analysis. With the described method various SAA isoforms can be isolated, purified and separated directly from human plasma/serum without prior ultracentrifugation.     

Secretion of lipid-poor nascent human apolipoprotein apoAI, apoCIII, and apoE by cell clones expressing the corresponding genes

The human apolipoprotein apoAI, apoCIII, and apoE genes were placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus vector that also contained the human metallothionein 1A gene. Following transfection of mouse C127 cells with the expression vector, cell clones resistant to Cd2+ were selected and found to express in high abundance specific apolipoprotein genes. Individual cell clones expressing apoAI, apoCIII, or apoE genes were used further to study the isoprotein composition and the flotation properties of the corresponding nascent apolipoproteins. It was found that the lipoproteins secreted by cell clones expressing the apoAI, apoCIII, and apoE genes consisted of the proapoAI disialylated form of apoCIII (apoCIIIS2) and mainly sialylated forms of apoE. Separation of the secreted apolipoproteins by density gradient ultracentrifugation resulted in limited flotation of nascent apoAI, apoE and apoCIII in the high density lipoprotein (HDL) fraction. Similar analysis in the presence of human serum increased the flotation of apoAI, apoE, and apoCIII to 6.5-, 4.5-, and 5.5-fold, respectively, and resulted in their redistribution to various lipoprotein fractions. HDL increased the flotation of apoAI to 12-fold and very low density lipoprotein (VLDL) increased the flotation of apoCIII and apoE to 6.5- and 5.5-fold, respectively. These findings suggest that in the cell system used, the majority of nascent apoAI, apoCIII and apoE is secreted in the lipid-poor form, which then associates extracellularly with preexisting lipoproteins.     

THE PLASMA DISTRIBUTION OF BENZOPORPHYRIN DERIVATIVE and THE EFFECTS OF PLASMA LIPOPROTEINS ON ITS BIODISTRIBUTION

The plasma distribution and biodistribution of benzoporphyrin derivative were examined. Two analogs of benzoporphyrin derivative were mixed with human plasma in vitro and recovered in the lipoprotein fractions upon separation by chromatography or ultracentrifugation. The majority of both analogs was recovered with high density lipoprotein. The effect of prebinding benzoporphyrin derivative to lipoproteins on the biodistribution of the drug in vivo was studied in tumor bearing DBA/2J mice. At 3, 8 and 24 h post-injection, tumor and tissue samples were excised and analyzed for benzoporphyrin derivative content. Precomplexing benzoporphyrin derivative with low density lipoprotein or high density lipoprotein led to significantly (P less than 0.05) greater tumor accumulation than in aqueous solution.     

Schnelle Dichtegradienten-Zentrifugation dispergierter Teilchen

Zusammenfassung Zur Untersuchung der chemischen Zusammensetzung dispergierter Teilchen, z. B. in Polymerlatices, wird eine schnelle Methode der Dichtegradienten-Zentrifugation entwickelt. Mit ihr kann die Verteilung der Dichte und damit der chemischen Zusammensetzung der Teilchen in wenigen Minuten bestimmt werden. Die bisher dafür verwendete konventionelle Dichtegradienten-Zentrifugation erfordert Versuchszeiten von 15 bis 30 Stunden.Das Prinzip der schnellen Methode beruht auf der Überschichtung von chemisch weitgehend gleichen oder ähnlichen Dispersionsmitteln unterschiedlicher Dichte (z. B. H₂O und D₂O oder Buten-(3)-ol-(2) und Ethylenglykol) und der Einstellung des Dichtegradienten durch Diffusion. Hauptanwendungsgebiete sind die Ermittlung der chemischen Zusammensetzung und Uneinheitlichkeit von Co-, Pfropf- und Mischpolymerlatices und die Bestimmung von Teilchenvernetzungsgraden.

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Summary For the investigation of the chemical composition of dispersed particles, e. g. in polymer latices, a rapid method of density gradient centrifugation is developed. The method yields in a few minutes the density distribution and by this the distribution of the chemical composition of the particles. The standard density gradient centrifugation needs for the same results more than 15 hours.The rapid method is based on adding a layer of a dispersion medium of low density to a chemically similar medium of high density (e. g. H₂O to D₂O or 3-butene-2-ol to ethylene glycol) and forming the density gradient by diffusion. The main field of application is the investigation of the chemical composition and heterogeneity of copolymer latices and latex mixtures and of the particle cross linking.

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Mit 10 Abbildungen und 2 Tabellen     

Properties of DNA from Various Local Strains of Bradyrhizobium sp. (Vigna radiata)

The buoyant density of DNA from various local strains of mung-bean nodule bacteria (Bradyrhizobium sp.) was determined by ultracentrifugation in an equilibrium CsCl gradient. The number of GC base pairs (61.1-62.7% mol %) and DNA melting point (94.3-95.0°C) were calculated from the buoyant density. DNA preparations purified by ultracentrifugation in a CsCl gradient by electrophoresis show a bimodal distribution. The DNA composition varies little (1.6 mol %). The electrophoretic distribution of local and Type-strain mung-bean nodule bacteria are identical. This proves that they belong to a single taxonomic group.     

Lipoproteins modulate growth and differentiation of cultured human epidermal keratinocytes

The effects of various lipoproteins on the growth and the differentiation of cultured normal human keratinocytes were investigated. Primary cultures of human epidermal keratinocytes were obtained from neonatal foreskin, and then added with lipoproteins, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Cell growth potential was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. VLDL and LDL enhanced keratinocytes growth and LDL receptor expression at the plasma membrane level. These effects were more remarkably observed in cells cultured with VLDL than in cells cultured with LDL. Apolipoprotein E (ApoE) was highly increased in VLDL treated cells. These results suggest that VLDL binds with high affinity to cell surface receptors and induces cell proliferation.     

液相色谱-串联质谱法检测人血浆及脂蛋白唾液酸的含量

建立了液相色谱-串联质谱(LC-MS/MS)检测人血浆脂蛋白唾液酸的方法,并比较了糖尿病患者与健康人血浆脂蛋白唾液酸的差异。采用pH=2的醋酸水解唾液酸,高速离心后采用优化的条件进行LC-MS/MS分析。结果表明:在负离子模式下,唾液酸的检出限和定量限分别为7.4和24.5 pg。唾液酸在2.5~80 ng/mL范围内呈良好的线性关系,相关系数R²大于0.998。糖尿病患者(平均年龄51.6岁)和健康人(平均年龄50.7岁)血浆中唾液酸的含量分别为(548.3±88.9)和(415.3±55.5)mg/L;两组实验人群中极低密度脂蛋白、低密度脂蛋白和高密度脂蛋白唾液酸的含量分别为(4.91±0.19)、(6.95±0.28)、(3.61±0.22)μg/mg和(2.90±0.27)、(7.03±0.04)、(2.40±0.09)μg/mg。糖尿病患者极低密度脂蛋白和高密度脂蛋白唾液酸含量显著高于同龄健康人水平(P<0.01)。该法可快速检测血浆中脂蛋白唾液酸含量,省时省力。     

Analysis of lipoproteins using 2D diffusion-edited NMR spectroscopy and multi-way chemometrics

This study represents the first application of multi-way calibration by N-PLS and multi-way curve resolution by PARAFAC to 2D diffusion-edited ¹H NMR spectra. The aim of the analysis was to evaluate the potential for quantification of lipoprotein main- and subfractions in human plasma samples. Multi-way N-PLS calibrations relating the methyl and methylene peaks of lipoprotein lipids to concentrations of the four main lipoprotein fractions as well as 11 subfractions were developed with high correlations (R = 0.75-0.98). Furthermore, a PARAFAC model with four chemically meaningful components was calculated from the 2D diffusion-edited spectra of the methylene peak of lipids. Although the four extracted PARAFAC components represent molecules of sizes that correspond to the four main fractions of lipoproteins, the corresponding concentrations of the four PARAFAC components proved not to be correlated to the reference concentrations of these four fractions in the plasma samples as determined by ultracentrifugation. These results indicate that NMR provides complementary information on the classification of lipoprotein fractions compared to ultracentrifugation.     

Abtrennung von Radionukliden in der Gasphase

Zusammenfassung Die Abtrennung von Radionukliden in der Gasphase spielt bei den verschiedensten Problemen eine Rolle. In der vorliegenden Arbeit werden Beispiele gegeben für Abtrennungen von sehr geringen Mengen, wobei ein Vergleich der Nachweisempfindlichkeiten für On-line und Off-line Gas-Chromatographie durchgeführt wird. Es werden Beispiele angegeben für die Trennung durch Adsorptions-Chromatographie im Temperaturgradienten, für mehrdimensionale Gas-Chromatographie mit verschiedenen Reaktivgasen und für die Trennung durch Komplexbildung mit Wandsubstanzen.

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Separation of radionuclides in the gas phase

The separation of radionuclides in the gas phase is important for very different problems. In this study examples are given for the separation of very small quantities and the analytical sensitivity of on-line and off-line gas chromatography is compared. Examples are described for the separation by adsorption chromatography in a temperature gradient, for multidimensional gas chromatography using different reactive gases and for the separation by complex formation with the wall material.

     

Direktbestimmung von Cu, Fe, Zn, Ca, Mg und Na im Serum mittels ICP-Emissionsspektralanalyse

Zusammenfassung Die Anwendung der ICP-Emissionsspektralanalyse für die direkte Bestimmung von Cu, Fe, Zn, Ca, Mg und Na in menschlichem und tierischem Blutserum wird beschrieben. Aufgrund der hohen Anregungstemperatur in einem Plasma treten dabei die von der konventionellen Atomabsorptionsspektroskopie her bekannten chemischen Interferenzen nicht auf, so daß mit geringen Probenmengen (max. 1 ml Serum für den Nachweis dieser 6 Elemente) ein sehr empfindlicher und sehr gut reproduzierbarer Nachweis möglich ist.

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Direct determination of Cu, Fe, Zn, Ca, Mg, and Na in serum by means of ICP-emission spectrometry

Summary The application of ICP-emission spectroscopy for the determination of Cu, Fe, Zn, Ca, Mg and Na in human and animal serum is described. Due to the very high excitation temperature in a plasma the well known chemical interferences in atomic absorption spectroscopy do not appear in this technique. It is possible to carry out very sensitive and very well reproducible measurements on very small amounts of the sample (the total sample consumption for the determination of the above mentioned six elements is less than 1 ml).

     

The determination of aluminium in human plasma

Summary Determination of aluminium in human plasma is of great interest in monitoring dialysis patients under oral aluminium therapy. Flameless atomic-absorption is chosen as the method because of the low normal levels of this non-essential trace element. A method avoiding the analytical problems of aluminium determination in human plasma is described. Normal values for healthy persons and levels for dialysis patients are given.

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Aluminiumbestimmung in menschlichem Plasma

Zusammenfassung Die Bestimmung von Aluminium im menschlichen Plasma ist von großem Interesse bei der Überwachung von Dialysepatienten, die unter oraler Aluminiumtherapie stehen. Die flammenlose Atomabsorption ist die Methode der Wahl, da die Normalwerte dieses nicht essentiellen Spurenelements sehr niedrig sind. Eine Methode, die die analytischen Probleme bei der Bestimmung von Aluminium im menschlichen Plasma vermeidet, wird beschrieben. Normalwerte gesunder Personen und solche von Dialysepatienten werden angegeben.

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Dedicated to the 60th birthday of Prof. Dr. G. Zigeuner.     

The preparative dynamic density gradient method

In this work the development of a new ultracentrifugation method is described. The preparative density gradient method (PDDG) allows the fractionation of colloidal systems according to the density of their components. The fractions—collected separately—can be subjected to further investigations with any appropriate physical or analytical method. The particle density of a colloidal system is a very valuable information that can be used in many ways. Here for the first time a method is presented that could make the access to that information much easier for laboratories throughout the world. The preparative dynamic density gradient is performed in a preparative ultracentrifugation tube by layering heavy water under a sample that is dispersed or dissolved in water. The water and heavy water molecules undergo a fast diffusion process finally leading to a homogeneous solvent mixture. While reaching this equilibrium the water–heavy water concentration varies over the cell radius. Thus a density gradient is built up. Fractions of the sample will move to the radial position where their densities are matched. They can then be collected separately. In this work basic experiments and results that lead to the development of the PDDG will be presented. Calculations based on basic ultracentrifugation theory confirm the described experimental findings. An example will demonstrate the problem solving capability of this new method.     

Tetraphenylporphyrins monosubstituted with glycerol diester units. Synthesis and characterization

Summary A novel 1-monoglyceryl ether of 5-(4-hydroxyphenyl)-10,15,20-tritolylporphyrin and related diacid esters with very high solubility in hydrophobic solvents were synthesized.

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Glycerindiester-monosubstituierte Tetraphenylporphyrine. Synthese und Charakterisierung

Zusammenfassung Es wurde ein neuer 1-Monoglycerylether von 5-(4-Hydroxyphenyl)-10,15,20-tritolylporphyrin und verwandte Disäuren-ester mit hoher Löslichkeit in hydrophoben Lösungsmitteln synthetisiert.

     

Feldverdampfung von Metallen und Legierungen bei tiefen und höheren Temperaturen

Zusammenfassung Die verschiedenen Interpretationen der Feldverdampfung von Metallen und Legierungen im Ultrahochvakuum und in Gegenwart von Gasen, u. zw. Helium, Neon, Wasserstoff, Sauerstoff und Stickstoff, wurden diskutiert. Experimentell ist bei der Feldverdampfung von Übergangsmetallen ein recht unterschiedliches Verhalten festzustellen, insbesondere hinsichtlich des Auftretens von Molekülionen aus Metall-Gas-Verbindungen.

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Field evaporation of metals and alloys at low and high temperatures

Summary The different interpretations of field evaporation of metals and alloys in ultra high vacuum and in the presence of gases such as helium, neon, hydrogen, oxygen and nitrogen are discussed. The behaviour of the transition metals in field evaporation is remarkably different as to the formation of ionized molecules of metal-gas compounds.

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Vorgetragen beim 10. Kolloquium über metallkundliche Analyse, Wien, 3.-5. November 1980. -Herrn Prof. Dr. Hanns Malissa zum 60. Geburtstag gewidmet.     

Diffuse reflection and polarization modulation FT-IR spectrometry of surface species

Summary Two techniques for measuring the infrared spectra of adsorbed species on surfaces of different types are described. Diffuse reflectance spectra of species adsorbed on powdered substrates with a high surface area can be measured with an exceptionally high signal-to-noise ratio. The technique is illustrated by the adsorption of carbon monoxide on rhodium supported on alumina at metal loadings between 0.1 and 10%. Some doubt is cast on previous band assignments for adsorbed CO molecules. Grazing incidence reflection-absorption spectrometry can be used to measure the spectra of molecules on flat metallic substrates of very low surface area. Attempts to develop a technique for observing the intermolecular vibration between the adsorbate and the substrate using a unique triple modulation approach are discussed.

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FT-IR-Spektrometrie mit diffuser Reflexions- und Polari-sations-Modulation von Adsorbaten an Oberflächen

Zusammenfassung Zwei Techniken zur Messung von Infrarot-Spektren von Adsorbaten verschiedener Art werden beschrieben. Diffuse Reflexionsspektren von Adsorbaten an pulverförmigen Substraten mit einer großen Oberfläche können mit einem außerordentlich großen Signal-zu-RauschVerhältnis gemessen werden. Als Beispiel dient die Adsorption von Kohlenstoffmonoxid an Rhodium auf Aluminiumoxid bei Metallgehalten von 0,1 bis 10%. Einige Zweifel an früheren Bandzuweisungen für adsorbierte CO-Moleküle werden geäußert. Reflexions-Absorptions-Spektrometrie mit streifendem Einfall kann zur Messung der Spektren von Molekülen an flachen metallischen Substraten mit sehr geringer Oberfläche benutzt werden. Versuche zur Entwicklung einer Technik für die Beobachtung der intermolekularen Schwingungen zwischen dem Adsorbaten und dem Substrat mittels einer einzigartigen Dreifach-Modulationstechnik werden diskutiert.

     

Probleme der Analytik von Pyridin-Nucleotid-Coenzymen

Zusammenfassung Die Qualitätsbestimmung von Pyridin-Nucleotid-Coenzympräparaten mit den üblichen Analysenmethoden, wie enzymatische Bestimmung und Extinktionsmessungen, ist aufgrund der relativ großen Fehlerbreite dieser Methoden schwierig. Ein spezielles Problem stellt die quantitative Erfassung UV-absorbierender Begleitstoffe dar. Ein neu entwickeltes Chromatographie-Verfahren erlaubt, diese Verunreinigungen abzutrennen und bis zu einem Gehalt von 0,1% zu bestimmen. Am Beispiel von NAD wird die Methode näher erläutert.

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Problems of the analysis of pyridine-nucleotide coenzymesI. -nicotinamide-adenine-dinudeotide

The quality of pyridine-nucleotide preparations is very difficult to evaluate by the common analytical methods like enzymatic procedures and measurements of the optical density because of the relatively high error of these methods. It is especially difficult to obtain exact results for the content of UV-absorbing compounds. A new procedure is described by which these components can be separated chromatographically and estimated down to a concentration of 0.1%. The results with NAD are discussed in detail.

     

Serum amyloid A protein in very low density--and high density lipoproteins during the course of acute myocardial infarction

In a preceding paper we reported on the detection and characterization of human serum amyloid A protein (SAA) in very low density lipoproteins (VLDL) and high density lipoproteins (HDL) of patients after acute myocardial infarction. Here we describe the time course of the occurrence of SAA in VLDL and HDL in the postinfarction period. SAA reached its maximum in VLDL and HDL approximately 53 h after the acute event. At the peak of the acute-phase response, SAA comprised as much as 38% of the total apoproteins of VLDL and HDL. SAA appeared at the same points in time and with nearly the same concentrations in VLDL and HDL. We conclude that SAA is not exchanged in plasma between lipoproteins of different densities and that this protein is secreted on its own by hepatocytes and not as a part of an already constituted lipoprotein particle.