RESUMEN
We describe a new method of immunoelectrophoresis with a continuous gradient polyacrylamide gel in the first dimension and an agarose-dextran gel in the second dimension with one or two layers of antibody. The use of a polyacrylamide gel in the first dimension allows better resolution of lipoproteins than with crossed immunoelectrophoresis using agarose gel in both dimensions. The use of two layers of antibody in the second dimension also enhances the specificity of characterization and the resolution of the separation. Thus, using a layer of anti-apo A-I combined with a layer of anti-apo A-II, three particles containing only apo A-I and three containing both apo A-I and A-II could be separated.
Asunto(s)
Inmunoelectroforesis/métodos , Lipoproteínas HDL/aislamiento & purificación , Adulto , Apolipoproteínas/química , Apolipoproteínas/aislamiento & purificación , Electroforesis en Gel Bidimensional/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Femenino , Humanos , Lipoproteínas HDL/química , EmbarazoRESUMEN
A modification of crossed immunoelectrophoresis for the analysis of plasma lipoproteins is described and is called polyacrylamide gel-crossed immunoelectrophoresis. The incorporation of albumin in the first-dimensional gel facilitates the transfer of the larger lipoproteins containing apolipoprotein B from the first-dimensional gel to the second dimension. Furthermore, under this condition the quantitation of total apolipoprotein B by polyacrylamide gel-crossed immunoelectrophoresis is in good agreement with the results obtained by rocket immunoelectrophoresis or nephelometry. The correlation between polyacrylamide gel-crossed immunoelectrophoresis and rocket immunoelectrophoresis is good for total apolipoprotein B (p greater than 0.001) and apolipoprotein A-I (p greater than 0.001). Polyacrylamide gel-crossed immunoelectrophoresis also offers interesting aspects to study the plasma lipoprotein classes and subclasses in different cases: normal plasma, current and complex dyslipoproteinemias in the presence or absence of lipoprotein small a. In a case of dyslipoproteinemia of Fredrickson's Type V polyacrylamide gel-crossed immunoelectrophoresis demonstrates the presence of a small-sized Lp (a) major peak in the low density lipoprotein (LDL) zone and of a large-sized Lp (a) minor peak in the very low density lipoprotein (VLDL) zone.
Asunto(s)
Electroforesis , Lipoproteínas/sangre , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Inmunoelectroforesis , Lipoproteínas/clasificación , MasculinoRESUMEN
Fluorescence quenching by iodide ions has been found to be higher in isolated Tangier low density lipoprotein (LDL2) than in isolated normal LDL2. Apolipoprotein (apo) B-100 is the main protein component of these lipoproteins and its tryptophanyl residues (Trp) are known to be the most hydrophobic and to be responsible for protein fluorescence. Trp exposure can thus be calculated; it was 0.50 in Tangier and 0.42 and 0.41 in insulin-dependent diabetics (IDD) and normal controls, respectively. The greater fluorescence quenching of Tangier LDL2 reveals a shallower embedding of Trp which is principally due to a lowered free cholesterol (FC) level in the shell and a smaller lipid core, itself dependent on a drop in cholesterol esters (CE). This is in accordance with the electrophoretic properties of Tangier LDL2 and suggests that Tangier LDL2 may be considered to be modified.
Asunto(s)
Apolipoproteínas B/sangre , Hipolipoproteinemias/sangre , Lipoproteínas LDL/sangre , Enfermedad de Tangier/sangre , Adulto , Apolipoproteína B-100 , Electroforesis de las Proteínas Sanguíneas , Colesterol/sangre , Ésteres del Colesterol/sangre , Fluorometría , Humanos , Masculino , Conformación Proteica , Triglicéridos/sangreRESUMEN
Fluorescent triacylglycerols containing pyrenedecanoic (P10) and pyrenebutanoic (P4) acids were synthesized and their hydrolysis by lipases from human gastric juice and stomach homogenate was investigated. The existence in stomach homogenate of four different lipolytic enzymes hydrolyzing fluorescent triacylglycerols is suggested by the comparison of various enzymatic properties: acyl chain length specificity, heat inactivation and effect of detergents (Triton X-100 and taurocholate), serum albumin, diethyl-para-nitrophenyl phosphate (E600) and other inhibitors. (1) The acid pH4-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol and exhibited the characteristic properties of the lysosomal lipase: the maximal activating effect of detergents occurs at relatively high concentrations (the substrate/detergent optimal molar ratios were 1:5 and 1:25 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively); its activity was strongly inhibited by para-chloromercuribenzoate (2.5 mmol/l), but was not significantly affected by serum albumin and E600 (10(-2) mmol/l). (2) The neutral pH7-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol. It is resistant to E600 and heat-stable, similarly to the acid pH4-lipase, but it is well discriminated from the acid enzyme by its substrate/detergent optimal molar ratios (1:2 and 1:3 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively), whereas higher detergent concentrations, optimal for the acid lipase, are strongly inhibitory for the neutral enzyme. (3) The pH5-lipase present in gastric juice as well as in stomach homogenate exhibited properties obviously discriminating it from the other lipolytic enzymes from stomach homogenate: broad substrate specificity for P10- as well as P4-triacylglycerols, activation by low concentrations of amphiphiles (with optimal ratios triacylglycerols/taurocholate, triacylglycerols/taurocholate and triacylglycerols/phosphatidylcholine around 1:1, 1:3 and 1:0.1, respectively), heat-lability, strong activation by serum albumin and inhibition by E600 (10(-2) mmol/l). This pH5-lipase is the sole lipolytic enzyme present in gastric juice and is probably identical with the well-known 'gastric' lipase. (4) A pH7.5-enzyme is characterized by its specificity for P4-triacylglycerols, its heat-lability at 50 degrees C and its strong inhibition by E600 (10(-2) mmol/l).
Asunto(s)
Jugo Gástrico/enzimología , Lipasa/metabolismo , Estómago/enzimología , Estabilidad de Enzimas , Colorantes Fluorescentes , Humanos , Hidrólisis , Cinética , Pirenos , Especificidad por Sustrato , Termodinámica , TriglicéridosRESUMEN
A fluorescent radiolabeled triacylglycerol has been synthesized by using a fluorescent fatty acid (pyrene decanoic acid) and a radiolabeled oleic acid. This analog of the natural substrate, 1(3)pyrene decanoic-2,3 (1,2)-dioleoyl-sn-glycerol, has been tested as substrate for determining lipoprotein lipase and hepatic triacylglycerol lipase activities in post-heparin plasma. Optimal conditions for the determination of the two post-heparin plasma lipases were similar to those using radiolabeled triolein. Using this substrate, both post-heparin lipases exhibited their characteristic properties (pH optimum and effect of inhibitors) and attacked external ester bonds (1 or 3) containing pyrene decanoic and oleic acids at a similar rate.
Asunto(s)
Lipasa/sangre , Lipoproteína Lipasa/sangre , Hígado/enzimología , Triglicéridos , Animales , Concentración de Iones de Hidrógeno , Hidrólisis , Lipasa/antagonistas & inhibidores , Lipoproteína Lipasa/antagonistas & inhibidores , Conejos , Cloruro de Sodio/farmacología , Dodecil Sulfato de Sodio/farmacología , Espectrometría de Fluorescencia , TritioRESUMEN
1. Synthetic cholesteryl esters with various acyl chain length (C2-C18) are hydrolysed by several enzymes in hamster liver. 2. The comparison of effect of inhibitors, divalent cations, detergents, pH and substrate specificity allows discrimination between four enzymes hydrolyzing cholesteryl esters, which are characterized by their enzymatic properties, two cholesterol esterases (resistant to E600) hydrolyzed medium- and long-chain cholesteryl esters, whereas short-chain cholesteryl esters were hydrolyzed by two different carboxylesterases (dramatically inhibited by E600). 3. The acid cholesterol esterase (identical to the lysosomal lipase) exhibited a pH optimum at pH 5.0 and is activated by 1 mM taurocholate. 4. The alkaline cholesterol esterase (pH optimum 7.5) is not very sensitive to the tested effectors. 5. Both acid and alkaline carboxylesterases (pH optima 5.5 and 7.5), were characterized by their strict dependence on divalent cations (Mn2+ or Mg2+). 6. The acid carboxylesterase was inhibited by increasing concentrations of Triton X-100, whereas the alkaline carboxylesterase was dramatically activated by 2 g/l Triton X-100. 7. No significant difference was observed in activities of cholesterol esterases or carboxylesterases between normal and FEC hamster livers.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Hipercolesterolemia/enzimología , Hígado/enzimología , Esterol Esterasa/metabolismo , Animales , Carboxilesterasa , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Cationes , Cricetinae , Concentración de Iones de Hidrógeno , Masculino , Mesocricetus , Paraoxon/farmacología , Esterol Esterasa/antagonistas & inhibidores , Tensoactivos/farmacologíaRESUMEN
Cholesteryl esters with various chain lengths of fatty acid, radioactive (C2-C18:1) and fluorescent (pyrene butanoic and decanoic acid, P4 and P10, respectively) were synthesized and their hydrolysis was investigated in lymphoid cell lines from normal subjects and from Wolman's disease patients. The comparison of their hydrolysis showed that three cholesterol esterases were present in normal lymphoid cell lines: the first, active at pH 4.0, hydrolysed preferentially cholesteryl esters of acyl chain length more than 8 carbons, and P10-cholesteryl ester. This acid cholesterol esterase, strongly inhibited by SH-blocking agents and resistant to E600, was severely deficient in Wolman lymphoid cell lines and corresponded to acid lysosomal lipase. The second and the third cholesterol esterases, active at pH 6.0 and 8.0, respectively, hydrolysed shorter-chain derivatives: the pH 8.0 enzyme was specific for short-chain derivatives (cholesteryl acetate, butyrate and P4), whereas the pH 6.0 activity showed a broader specificity, since it hydrolysed all the cholesteryl esters, with a maximum of activity on cholesteryl acetate and butyrate. The pH 6.0 and 8.0 enzymes were heat-labile, inhibited by E600, resistant to SH-blocking agents and not deficient in Wolman lymphoid cell lines. The hypothetical physiological role of these enzymes is discussed.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Isoenzimas/metabolismo , Linfocitos/enzimología , Esterol Esterasa/metabolismo , Xantomatosis/enzimología , Línea Celular , Herpesvirus Humano 4 , Calor , Humanos , Concentración de Iones de Hidrógeno , Micelas , Relación Estructura-Actividad , Especificidad por Sustrato , Ácido Taurocólico/farmacología , Xantomatosis/sangreRESUMEN
Lipid analyses of the bronchoalveolar washing fluid of three patients affected with chronic unidentified pneumonia showed the presence of a major hydrophobic compound identified as paraffin oil by thin layer chromatography, infrared spectroscopy and gas liquid chromatography (by comparison with various paraffin preparations as standards). Moreover the phospholipid and neutral lipid composition was determined. In conclusion, the usefulness of bronchoalveolar lavage and its biochemical analysis is discussed as a non-invasive method of diagnosis and therapeutic approach to liquid paraffin pneumonia (in the cases herein described, between 100 and 500 mg of paraffin oil were extracted from 100 ml of bronchoalveolar washing fluid.
Asunto(s)
Aceite Mineral/análisis , Neumonía por Aspiración/diagnóstico , Neumonía Lipoidea/diagnóstico , Alveolos Pulmonares/análisis , Anciano , Bronquios/análisis , Cromatografía de Gases , Cromatografía en Capa Delgada , Femenino , Humanos , Masculino , Persona de Mediana Edad , Espectrofotometría Infrarroja , Irrigación TerapéuticaRESUMEN
In order to evaluate the effect of hypercholesterolemia on the surface properties of low density lipoproteins (LDL), the quenching by iodide ions of the native fluorescence of human plasma LDL was studied on normolipidemic and hypercholesterolemic type IIa subjects. A significant difference (P less than 0.001) was found between these two groups (20 patients with type IIa hyperlipoproteinemia, 18 normolipidemic subjects). Furthermore, the fluorescence quenching (F0-F1)/F0 (F0 and F1 fluorescence intensity respectively in the absence and in the presence of iodide ions is negatively correlated with the relative LDL-cholesterol level (LDL-cholesterol/LDL-apoprotein). In contrast, this quenching is positively correlated with the relative LDL-non-apo-B level (LDL-non-apo-B/LDL apo). It is suggested that the greater the LDL-cholesterol level, the more embedded are the tryptophyl residues in the hydrophobic core. In contrast, the greater the LDL-non-apo-B level, the more exposed are the tryptophyl to the aqueous environment. Thus, a significant conformation change of the superficial apolipoproteins occurs, which could affect the immunological properties of the LDL and their affinity to the LDL receptors.
Asunto(s)
LDL-Colesterol/sangre , Hiperlipoproteinemia Tipo II/sangre , Lipoproteínas LDL/sangre , Fluorescencia , Humanos , Yodo , Conformación Proteica , TriptófanoRESUMEN
The chemical synthesis of bis(diacylglycero) phosphate previously named bisphosphatidic acid, starting with a diacylglycerol and phosphatidic acid, is described. The phosphodiester bond formation is catalyzed by triisopropylbenzenesulfonylchloride. This simple approach allows the preparation of saturated as well as unsaturated bis(diacylglycero)phosphate species in one step without the use of any protecting group. The methods used until now yield only mono-acid species, or mixed-acid unsaturated species after many steps involving the introduction and the removal of protecting groups. The synthetic products have been characterized by component analysis and NMR-techniques.
Asunto(s)
Ácidos Fosfatidicos/síntesis química , Catálisis , IsomerismoRESUMEN
The effect of an aqueous dispersion of succinylphosphatidylcholine on an aqueous suspension of phosphatidylcholine vesicles was studied by gel chromatography, freeze-fracture electron microscopy and proton nuclear magnetic resonance with Mn2+ (broadening paramagnetic reagent). Total phospholipid concentrations were in the range 10--20 mM. Succinylphosphatidylcholine is in micellar form and behaves as a detergent. The structures obtained depend on the molar percentage of succinylphosphatidylcholine. Above a succinylphosphatidylcholine molar percentage of 60%, mixed micelles are formed, assumed to be essentially spherical. Below a succinylphosphatidylcholine molar percentage of 30%, principally mixed vesicles are observed, with an external diameter of 215--240 A, and an almost constant internal volume. Between 30 and 60% of succinlyphosphatidylcholine, a mixture of these structures is obtained; rod-shaped profiles are also observed in electron microscopy, which may correspond to sections of leaky vesicles or to a new kind of cylindrical micelle.