Browsing by Author "Orozco, Enrique"

A relatively little time consuming simple method based on the treatment of bile with cholesterol oxidase and subsequent high performance liquid chromatography measurement of the 3-ketocholesterol produced in order to determine the level of the cholesterol concentration is described. The method avoids bilirubin interferences, has high reproducibility and recovery assays give 100 % values. It is highly sensiti\·e and suitable for use in the determination of cholesterol concentrations in bile and other bilirubin containing biological fluids.

Abstract~!. The effect of etofibrate, the ethandiol-1,2 diester of nicotinic and clofibric acids on bile production was studied in male rats that received a daily dose of 300 mg of etofibrate/kg body weight by stomach tube for 10 days and were compared with control rats receiving the medium. 2. The bile duct was cannulated, animals were intravenously given I μCi (4- 14C)-cholesterol/100 b.w. and bile was collected at different intervals for a total of 4 hr. 3. Etofibrate treatment decreased plasma cholesterol and triglyceride concentrations and increased the bile flow. The cummulative amount of both bile volume and total bile radioactivity secreted increased linearly in all the animals; the respective slopes being higher in etofibrate treated rats than in controls. 4. The main labelled component found in the bile was always bile acids rather than cholesterol and the proportion of each of these compounds was similar in both groups. Neither was any difference between the groups found in the concentration of bile acids, cholesterol and phospholipids nor in the cholesterol/(bile + phospholipid) ratio. 5. Besides other factors, the present results indicate that an increase in bile flow and biliary cholesterol excretion in its free form and after its conversion into bile acids should contribute to the hypocholesterolemic effect of elofibrate

Human total HDL (high-density lipoprotein), HDL2 and HDL3 were labelled in vitro by incubation with lipoproteindeficient serum (LPDS) which contained either [3H)cholesteryl oleate or [14C]cholesterol under different conditions. The lipoproteins were then subfractionated by heparin-Sepharose column chromatography, and three subfractions (A, Band C) were successively eluted from each preparation of HDL, HDL2 and HDL3• When the labelling was done at 37 °C for 17 h, the subfractions were homogeneously labelled with [3H]cholesteryl oleate. However, when it was performed for only 30 min at 4 °C, the subfractions showed marked differences in the 3H specific radioactivity, which was much higher in the C fractions than in the others. 2. 3H-labelled HDL2 and HDL3 subfractions behaved differently under the precipitant action of heparin-Mn2+; fraction C (the richest in apolipoprotein E) produced the largest amount of radioactive and chemical precipitate. More 3H radioactivity, but not the cholesterol, was precipitated from HDL2 or HDL3 by the reagent, demonstrating that 3H-labelled HDL2 and HDL3 behave like their fraction C, which becomes labelled to the highest specific radioactivity despite having the smallest mass. 3. The incubation of 3H-labelled HDL subfractions with human LPDS and very-low-density lipoprotein (VLDL) at 37 °C increased the quantity of 3H radioactivity that was precipitated, in proportion to the amount of VLDL present in the media. These changes were attributable to the action of cholesterol ester transfer protein, since they did not occur at 4 °C or when human LPDS was replaced with rat LPDS. 4. Kinetics of the transfer of HDL [3H]cholesteryl oleate to VLDL showed a greater apparent V for fractions A than for fractions B from either HDL2 or HDL3, whereas the apparent Km values were very similar, whi~h suggest that this transfer process is influenced by the apoprotein composition of the donor lipoprotein.

En esta revisión hemos intentado poner de manifiesto el papel que desempeña la LPL en el destino de los triglicéridos circulantes que, cuando son de origen endógeno (síntesis hepática de sustratos no-lipídicos y/o de los productos de la lipolisis, FFA y glicerol), lo hacen asociados a las VLDL. El efecto de distintos factores o situaciones fisiológicas sobre la actividad LPL varia de unos tejidos a otros. Así, mientras que en tejido adiposo la actividad de la enzima aumenta con la insulina y disminuye con el ayuno y la gestación, en corazón y músculo esquelético disminuye con la insulina y aumenta en situaciones de hipotrigliceridemia, con el ayuno, o por el tratamiento con glucocorticoides. En la glándula mamari3 la enzima es inducida por la prolactina, y éste es el mecanismo por el que aumenta su actividad al final de la gestación. En hígado de recién nacido, la LPL se relaciona de forma inversa con los niveles de insulina, mientras que aumenta con la hipotrigliceridemia, como ocurría en la enzima de corazón, y su actividad es inhibida tras el tratamiento con insulina. A pesar de estas diferencias en la respuesta a distintos efectores, la LPL parece ser igual des de el punto de vista estructural en todos los tejidos, y no conocemos a qué se deben esas diferencias funcionales. La LPL no solamente hidroliza a los triglicéridos que circulan en plasma asociados a las lipoproteínas ricas en ellos, quilomicrones y VLDL, sino que facilita la captación de los productos de dicha hidrólisis por el tejido subyacente, canalizando así la utilización de los mismos en el organismo. El destino metabólico de esos productos, FFA y glicerol, varía también de unos tejidos a otros. Mientras que en tejido adiposo son utilizados para su reesterificación y acúmulo, constituyendo una importante reserva energética para el organismo, en músculo cardíaco y esquelético y en hígado de recién nacido son oxidados para su utilización como fuente inmediata de energía; en glándula mamaria se transforman en los lípidos de la leche, y en pulmón son sustratos para la síntesis del tensioactivo. Así pues, la LPL juega un papel clave en el metabolismo de las VLDL y quilomicrones, tanto facilitando la transformación de estas lipoproteínas en otras de mayor densidad, que son finalmente eliminadas de la circulación, como modulando la canalización de 1011 triglicéridos que transportan a tejidos específicos, para su posterior metabolismo intracelular.