Analysis of Fatty Acid, and Lipoprotein, Metabolism by GC and HPLC: Effect of Low-Density Lipoprotein Apheresis

 Blood samples from 5 hyperlipidemic patients on chronic treatment with low-density lipoprotein (LDL) – apheresis were analysed for lipids and fatty acids in serum, lipoprotein fractions and erythrocyte membrane by capillary gas chromatography (GC), reversed-phase high-performance liquid chromatography (LC), spectrofluorometry and spectrophotometry. LDL-apheresis has been associated with significant changes of fatty acids metabolism in relation to triglyceride-rich lipoproteins. Oleic acid may exert its hypotriglyceridemic effect via VLDL, IDL, LDL and HDL fractions. Polyunsaturated fatty acids, associated with triglyceride metabolism via IDL or VLDL, are linoleic, gamma-linolenic and docosahexaenoic fatty acids. Received November 25, 1999. Revision September 5, 2000.     

Analytical capillary isotachophoresis: a routine technique for the analysis of lipoproteins and lipoprotein subfractions in whole serum

A capillary isotachophoretic separation technique was developed for lipoproteins in native serum which, compared with previous electrophoretic techniques, has negligible molecular sieve effects, does not need gel casting, is suitable for whole serum and has a high discriminative power for lipoprotein subfractions. The technique is based on pre-staining whole serum lipoproteins for 30 min at 4 degrees C before separation of 0.5 microliter of the sample in a free-flow capillary system (0.5 mm I.D.) with discontinuous buffer system. In normolipidaemic sera, high-density (HDL) and low-density lipoproteins (VLDL) are separated into two major subpopulations according to their net electric mobility. The identification of these fractions was confirmed by substitution with ultracentrifugally isolated lipoproteins and by their complete absence from Tangier and abetalipoproteinaemic serum. Triglyceride-rich very low-density lipoproteins (VLDL) revealed a defined zone between the HDL and LDL subpopulations. Our preliminary results indicate that the separation of human whole serum lipoproteins by capillary isotachophoresis is a promising method for the determination of lipoprotein subfractions.     

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。     

Über die Wirkung von Albumin auf die elektrophoretische Wanderungsgeschwindigkeit der menschlichen Serumlipoproteine auf Agarose-Gel

The electrophoretic mobility of the VLDL, LDL and HDL fractions increases on agarose gel after Sepharose 2 B gel filtration. This effect is broken of by albumin. The possibility of spontaneous hydrolysis of the triglycerides of the lipoproteins with a successive increase of the free fatty acid content and electric charge is discussed. The albumin effect is based on the binding of these free fatty acids.     

Isolation of plasma lipoproteins by a combination of differential and density gradient ultracentrifugation

Summary A method for preparative isolation of serum lipoproteins by a combination of differential and density gradient ultracentrifugation is presented. Total plasma lipoproteins are first isolated in a concentrated form by ultracentrifugation in a fixed angle rotor at a plasma background density of 1.21 kg/l. Subsequently, the various lipoprotein classes are separated by density gradient ultracentrifugation in a swinging bucket rotor. The procedure requires only two ultracentrifugation steps and combines advantages of both ultracentrifugation techniques.

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Isolierung von Plasmalipoproteinen durch eine Kombination von Differential- und Dichtegradient-Ultrazentrifugation

Abbreviations VLDL very low density lipoproteins - LDL low density lipoproteins - HDL high density lipoproteins - VHDL very high density lipoproteins     

Myocardial infarction patients show altered lipoprotein properties and functions when compared with stable angina pectoris patients

Several parameters and risk factors were compared between Korean male myocardial infarction (MI) patients (n = 10) and angina pectoris (AP) patients (n = 17) to search unique biomarkers for myocardial infarction (MI) in lipoprotein level. Individual serum and lipoprotein fractions (VLDL, LDL, HDL₂, HDL₃) were isolated and analyzed by lipid and protein determination and enzyme assay. The MI group was found to have a 25 and 30% higher serum cholesterol and triacylglycerol (TG) level than the AP group, respectively, however, their body mass index (BMI), LDL-cholesterol (C), HDL-C, and glucose levels fell within the normal range. MI patients were found to have an approximately two-fold higher level of serum IL-6 and an 18% lower serum apoA-I level than that of the AP group. LDL and HDL₂ fraction of the MI group were more enriched with TG than those of AP group. The increased TG was correlated well with the increased level of apoC-III in the same fraction. Cholesteryl ester transfer protein (CETP) activity and protein level were greatly increased in MI patients in the LDL and HDL₃ fractions. MI patients showed more severely oxidized LDL fraction than patients in the AP group, as well as the weakest antioxidant ability of serum. Conclusively, MI patients were found to have unique serum and lipoprotein characteristics including increased IL-6 and TG in serum, with CETP and apoC-III in the LDL and HDL fractions, as well as severely impaired antioxidant ability of HDL.     

CELLULAR DELIVERY AND RETENTION OF PHOTOFRIN: III. ROLE OF PLASMA PROTEINS IN PHOTOSENSITIZER CLEARANCE FROM CELLS

Abstract— Human plasma proteins, albumin, globulins and low density (LDL), high density (HDL) and very low density (VLDL) lipoproteins were tested for their effects on retention of Photofrin and three other photosensitizers in cultured cells. This was assessed by incubating the cells, subsequent to the exposure to Photofrin, in the photo-sensitizer-free medium containing various concentrations of different plasma proteins. Photofrin clearance levels differed with individual plasma proteins and also were dependent on concentration of these proteins in the incubation medium. All of the proteins except VLDL promoted clearance of Photofrin taken up by the cells in the presence of 5% human serum. Subsequent to some Photofrin exposure conditions (in the presence of 5% fetal bovine serum, or in protein-free medium), albumin, in contrast to LDL, HDL and globulins, exhibited decreased capacity for promoting the photosensitizer clearance from the cells. The VLDL showed very little or no effect in promoting cellular clearance of Photofrin, tetraphenyl porphine tetrasulfonate (TPPS₄), and di- and tetrasulfonated chloroaluminum phthalocy-anine (AlPcS₂ and AlPcS₄, respectively). The LDL seem to be particularly effective in promoting clearance of Photofrin and AlPcS₂ from the cells, whereas albumin and globulins were shown to be more effective than LDL and HDL in promoting the cellular clearance of TPPS₄.     

Charakterisierung von menschlichen Serumlipoproteinen mittels Gelfiltration auf Agarose (Sepharose 2 B)

Zusammenfassung Reine Lipoproteinfraktionen des menschlichen Serums werden durch Gelfiltration auf Sepharose 2 B charakterisiert. Die SäulenkonstantenKd für die Chylomicronen, VLDL, LDL und HDL werden zu den Dichteklassen dieser Lipoproteine in Beziehung gesetzt. Die Befunde an den Reinfraktionen werden mit den Elutionsmustern des menschlichen Normalserums und des Serums bei Patienten mit einer Hyperlipoproteinämie vom Typ III mit einem abnormen-Lipoprotein verglichen. Die diagnostische Bedeutung der Sepharose 2 B-Gelfiltration für die Erkennung der Hyperlipoproteinämien wird diskutiert.

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Characterisation of human serum lipoproteins by gel filtration on Agarose (Sepharose 2 B)

The constantKd for each fraction (chylomicrons, VLDL, LDL, HDL) is correlated to its density. The results with these pure fractions are compared with eluation patterns of normal serum and serum with hyperlipoproteinemia type III containing an abnormal-lipoprotein. The importance of agarose gel filtration for the diagnosis of hyperlipoproteinemias is discussed.

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2%ige Agarosepräparation der Fa. Pharmacia Fine Chemicals, Uppsala, Schweden.     

Profiling of phospholipids in lipoproteins by multiplexed hollow fiber flow field-flow fractionation and nanoflow liquid chromatography–tandem mass spectrometry

This study describes a coupled analytical method to carry out the systematic profiling of phospholipids (PLs) in high-density lipoproteins (HDL) and low-density lipoproteins (LDL) from human blood plasma. HDL and LDL of healthy human plasma samples were separated by size and collected on a semi-preparative scale using multiplexed hollow fiber flow field-flow fractionation (MxHF5). Phospholipid mixtures contained in the resulting HDL and LDL fractions were analyzed by shotgun nanoflow liquid chromatography–tandem mass spectrometry (nLC–ESI-MS–MS). We utilized a dual scan method for the separation and simultaneous characterization of complicated PL mixtures by nLC–ESI-MS–MS, such that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules were detected in positive ion mode in a first LC run. In a second LC run, phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA) were detected in negative ion mode. In this study, a total of 56 PLs from HDL and 52 PLs from LDL particles were characterized by their molecular structures from data dependent collision-induced dissociation (CID) experiments, and their relative abundances were compared.     

Electrophoretic behavior and partial characterization of disease-associated serum forms of gamma-glutamyltransferase

We have recently devised an improved procedure for the rapid electrophoretic separation of multiple forms of serum gamma-glutamyltransferase (GGT). This procedure is based on the separation on cellulose acetate strips, usually employed for lipoprotein electrophoresis, followed by visualization with a fluorescent reagent. The method is highly sensitive and the fractions are more clearly resolved than with other procedures. Reference intervals have been evaluated in the sera from 142 healthy subjects and the patterns (two GGT forms comigrating with alpha 1 and alpha 2-globulin) are reproducible. In 150 sera from patients with various hepatobiliary diseases (including neoplasias), acute pancreatitis and non liver-involving neoplasias, we observed some disease-specific GGT forms: an albumin comigrating enzyme (Alb-GGT) specific of liver neoplasia; a gamma-globulin comigrating GGT (gamma-GGT) and a nonmigrating isoform (dep-GGT) both specifically associated to extrahepatic jaundice. Multiple lipoprotein fraction precipitation showed that beta-, gamma- and dep-GGT are complexes between GGT and low density lipoprotein and very low density lipoproteins (LDL + VLDL), and that some of the alpha 1-GGT from cirrhotic patients is a complex between GGT and high density lipoprotein (HDL). GGT fractions from normal subjects and Alb-GGT from patients with liver neoplasia do not appear to be complexed with lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photochemistry of Bacteriochlorophylls in Human Blood Plasma: 1. Pigment Stability and Light-induced Modifications of Lipoproteins

Transmetalated derivatives of bacteriochlorophyll are promising sensitizers in photodynamic therapy. Protocols using short delay times between injection and irradiation cause interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the photochemistry of Zn- and Pd-bacteriopheophorbide (WST09), and of the highly polar taurinated Pd-derivative, WST11, in isolated fractions of human blood plasma. The stability of all pigments is increased in blood plasma, compared with monomeric solutions. Pd-bacteriopheophorbide is much more stable than the other two derivatives. It also has a higher capacity for inducing reactive oxygen species, yet the consumption of oxygen is comparable. There is furthermore evidence for photobleaching under anoxic conditions. The generation of hydroperoxides (ROOH) is faster with Pd- than with Zn-complexes; the formation of endoperoxides (ROOR′), measured as thiobarbituric acid reactive substances, is comparable with the two central metals. Formation of both ROOH and ROOR′ is increased in low-density lipoproteins (LDL) compared with high-density lipoproteins (HDL), which is probably related to the higher concentration of target molecules in the former. In HDL, extensive cross-linking is induced among the apolipoproteins; judged from the electrophoretic mobility of LDL and HDL particles, there is also a gross structural change. Photosensitized cross-linking is much less pronounced with high-density proteins.     

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.     

Erythrocyte spectrin alteration induced by low-density lipoprotein.

Addition of human plasma low-density lipoproteins (LDL) to intact human erythrocytes induces the erythrocytes to undergo morphologic transition from biconcave disks to echinocytes and spherocytes. The transformation is time-dependent. Two hours are required before echinocytes are detected by scanning electron microscopy. After two hours, LDL also decrease the phosphate content of spectrin by 40% relative to the control, suggesting that these lipoproteins modulate cell shape by influencing phosphorylation-dephosphorylation of a membrane-associated cytoskeletal protein. LDL do not induce depletion of intracellular adenosine triphosphate (ATP), nor do they inhibit cyclic adenosine monophosphate-independent protein kinases which phosphorylate spectrin. LDL stimulate membrane-bound phosphatases by a factor of two, thereby reducing the amount of phosphate covalently bound to membrane proteins. The observed effects are specific for LDL. High-density lipoproteins (HDL) do not stimulate dephosphorylation of spectrin or alter erythrocyte morphology. However, HDL protect the erythrocytes against LDL-induced alterations. These data suggest that the circulating lipoproteins have a role in maintaining erythrocyte morphology by regulating the extent of phosphorylation of spectrin.     

Simultaneous Determination of High-Density Lipoprotein,Very Low-Density Lipoprotein and Low-Density Lipoprotein Subclass in Human Serum by Microchip CE

Lipoproteins, especially high-density lipoproteins (HDL), very low-density lipoproteins (VLDL) and small, dense low-density lipoprotein (sdLDL), are believed to play an important role in the development of atherosclerosis. In this work, a simple, selective and sensitive method for the simultaneous monitoring of these lipoproteins in human serum using microchip capillary electrophoresis was developed. Gold nanoparticles were used as an additive to the running buffer to obtain the absolute separation of the lipoproteins. Under optimised conditions, the linear ranges of large buoyant low-density lipoproteins, sdLDL, VLDL and HDL were 10–800, 10–800, 40–1,000 and 20–800 μg L⁻¹, and their limits of detection were 5, 5, 15 and 8 μg L⁻¹, respectively. The intraassay and interassay relative standard deviation of lipoprotein peak areas were in the range of 3.8–7.4%. For practical application, variations in the serum lipoprotein of coronary heart disease patients were monitored by microchip-based CE. The results showed that the method was applicable for routine clinical use and allowed the rapid detection of different lipoprotein classes as well as their subclasses, thus greatly improving the analysis of atherosclerotic risk factors.

     

Anomalous change of viscosity and conductivity in blood plasma lipoproteins in the physiological temperature range

Study on temperature dependencies of viscosity (η) and conductivity (σ) of blood lipoproteins [high‐density lipoprotein (HDL), low‐density lipoprotein (LDL), and very low density lipoprotein (VLDL)] and A‐I apolipoprotein (human and rats) has revealed the presence of the anomalous region at temperature 35–38±0.5°C (T_c). Transition width is 2°C. Viscous flow enthalpy, activation energy (ΔH), and transition enthalpy (ΔH_trans.) as well as thermal coefficients Δη/ΔT and Δσ/ΔT on either side of T_c have been calculated. Transition heat is very low in human HDL, VLDL and apoA‐I, and in LDL it is higher by a factor of 4–5. Some mechanisms of the cortisol interaction with HDL and apoA‐I have been studied by infrared (IR) spectroscopy and conductometry. The hormone has been found to strengthen the tangle → α‐helixes and tangle → β‐structures transitions and increase the ordering of lipids. Therewith, ΔH_trans. rises markedly (13 and more times), and at the same time the anomalous region is shifted by 1–2°C in apoA‐I. The anomalous change of viscosity and conductivity in the physiological temperature range for all lipoprotein fractions and apoA‐I seems to be due to the structural phase transition in both proteins and lipids. In view of the heat capacity jump and a low value of ΔH_trans. in human HDL, one may assume the phospholipids of these particles to exhibit the orientation transition of smectic A↔C type, which is assigned to the second type of phase transition. The structural transition into apoA‐I is likely to contain the elements of phase transition of the first and second types. In human and rat VLDL, the smectic → cholesteric phase transition seems to occur. Enthalpy of viscous flow and structural transition in VLDL is higher for rats than for human. The pH shift of the medium to the neutral region (pH 6.1) results in shifting the anomalous region by 1–2°C. © 2001 John Wiley & Sons, Inc. Int J Quant Chem 81: 348–369, 2001     

Simultaneous Determination of High-Density Lipoprotein, Very Low-Density Lipoprotein and Low-Density Lipoprotein Subclass in Human Serum by Microchip CE

Lipoproteins, especially high-density lipoproteins (HDL), very low-density lipoproteins (VLDL) and small, dense low-density lipoprotein (sdLDL), are believed to play an important role in the development of atherosclerosis. In this work, a simple, selective and sensitive method for the simultaneous monitoring of these lipoproteins in human serum using microchip capillary electrophoresis was developed. Gold nanoparticles were used as an additive to the running buffer to obtain the absolute separation of the lipoproteins. Under optimised conditions, the linear ranges of large buoyant low-density lipoproteins, sdLDL, VLDL and HDL were 10?C800, 10?C800, 40?C1,000 and 20?C800 ??g L^?1, and their limits of detection were 5, 5, 15 and 8 ??g L^?1, respectively. The intraassay and interassay relative standard deviation of lipoprotein peak areas were in the range of 3.8?C7.4%. For practical application, variations in the serum lipoprotein of coronary heart disease patients were monitored by microchip-based CE. The results showed that the method was applicable for routine clinical use and allowed the rapid detection of different lipoprotein classes as well as their subclasses, thus greatly improving the analysis of atherosclerotic risk factors.     

Distribution of Chlorophyll- and Bacteriochlorophyll-derived Photosensitizers in Human Blood Plasma

Chlorophyll a and, in particular, bacteriochlorophyll a derivatives are promising candidates for photosensitizers in photodynamic therapy. The distribution of 21 (bacterio)chlorophyll derivatives among human blood plasma fractions was studied by iodixanol gradient ultracentrifugation and in situ absorption spectroscopy. Modifications of the natural pigments involved the central metal (Mg²⁺, Zn²⁺, Pd²⁺, none), the isocyclic ring (closed, open and taurinated), substituents at C-3 (vinyl, acetyl, 1-hydroxyethyl) and C-17³ (phytyl ester, free acid). Cellular blood components bound only a small fraction of the pigments. Distribution among low-density lipoproteins (LDL), high-density lipoproteins (HDL) and high-density proteins (HDP) of the plasma was influenced as follows: (1) application in Cremophor^® EL slightly altered pigment distribution by lipoprotein modification, (2) only very polar pigments with multiple hydrophilic substituents showed substantial HDP binding, (3) the presence of the esterifying alcohol at C-17³ caused enrichment in LDL, this was more pronounced with bacteriochlorophylls than with chlorophylls, (4) substituents at C-3 had only little influence on the distribution, (5) Zn²⁺-complexes were enriched in HDL compared to Mg²⁺ and Pd²⁺ complexes, indicating specific binding of the former. Equilibration of pigments among the different fractions was largely complete within 3 h.     

Characterization of plasma apolipoproteins by capillary electrophoresis.

The main apolipoproteins of plasma high-density lipoproteins (HDL) and low-density lipoproteins (LDL) were analyzed by capillary electrophoresis. Where possible the results were compared with slab sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Addition of the detergent SDS to the running buffer was essential for separation. Separations were carried out in bare silica and polyacrylamide-coated capillaries. The main apolipoproteins of HDL could be separated in an uncoated capillary filled with borax buffer containing 0.1% SDS. Using the coated capillary, a mixture of HDL and LDL apolipoproteins was resolved in less than 12 min. These preliminary studies indicate that capillary electrophoresis is a promising technique for screening plasma apolipoproteins.