Plasma membrane calcium pump activity in rat pancreatic islets: an accurate method to measure its calcium-dependent modulation.

The aim of this study was to quantify the glucose modulation of the plasma membrane calcium pump (PMCA) function in rat pancreatic islets. Ca2+-ATPase activity and levels of phosphorylated PMCA intermediates both transiently declined to a minimum in response to stimulation by glucose. Strictly dependent on Ca2+ concentration, this inhibitory effect was fully expressed at physiological concentrations of the cation (less than 0.5 muM), then progressively diminished at higher concentrations. These results, together with those previously reported on the effects of insulin secretagogues and blockers on the activity, expression and cellular distribution of the PMCA, support the concept that the PMCA plays a key role in the regulation of Ca2+ signaling and insulin secretion in pancreatic islets.

Óxidos de colesterol en langostinos frescos y congelados, crudos y a la plancha / Cholesterol oxidation products in fresh and frozen shrimps, raw and grilled

Los óxidos de colesterol (COPs) se relacionan con diferentes efectos tóxicos entre los que destacan su implicación en los procesos de aterosclerosis. Se estudió la presencia de óxidos de colesterol en langostinos comercializados en fresco y en congelación, tanto en crudo, como sometidos a una tecnología culinaria habitual (plancha). La determinación se realizó por cromatografía de gases acoplada a espectrometría de masas (CG-EM). En los langostinos frescos se detectaron todos los COPs analizados con excepción del 7 α-hidroxicolesterol, presentando una cantidad total de 33,15 µg COPs/g grasa. Por el contrario, en los langostinos comercializados congelados sólo se detectaron el 7-ketocolesterol y el 7ß-hidroxicolesterol, dando lugar a una cantidad total de 2,38 µg COPs/g grasa. Estos resultados indican la gran efectividad de la comercialización bajo condiciones de congelación de este tipo de alimentos en cuanto a ralentizar la formación de COPs. El tratamiento culinario incrementó el contenido de COPs en ambos tipos de langostinos, alcanzando 55,43 µg COPs/g grasa en los frescos y sólo 13,06 µg COPs/g grasa en los congelados (AU)

Cholesterol oxidation products (COPs) have been related to different toxic effects, being the atherosclerotic process one of the best known. The presence of cholesterol oxides in freshly and frozenly commercialised shrimps, both raw and grilled, was studied. The determination was made by gas chromatography-mass spectrometry (GCMS). Fresh shrimps showed significant amounts of all analysed COPs, except for 7α-hydroxycholesterol, accounting in total for 33.15 µg COPs/g fat. In contrast, in frozen commercialised shrimps only 7-ketocholesterol and 7ß-hydroxycholesterol were detected. These results point out the great effectiveness of the commercialisation of this type of products under freezing, in terms of to the minimisation of the COPs formation. The cooking method (grilling) increased the COPs content in both types of shrimps, reaching 55.43 µg COPs/g fat in fresh shrimps and only 13.06 µg COPs/g fat in frozen ones (AU)

[Cholesterol oxidation products in fresh and frozen shrimps, raw and grilled]. / Oxidos de colesterol en langostinos frescos y congelados, crudos y a la plancha.

Cholesterol oxidation products (COPs) have been related to different toxic effects, being the atherosclerotic process one of the best known. The presence of cholesterol oxides in freshly and frozenly commercialised shrimps, both raw and grilled, was studied. The determination was made by gas chromatography-mass spectrometry (GCMS). Fresh shrimps showed significant amounts of all analysed COPs, except for 7alpha-hydroxycholesterol, accounting in total for 33.15 microg COPs/g fat. In contrast, in frozen commercialised shrimps only 7-ketocholesterol and 7beta-hydroxycholesterol were detected. These results point out the great effectiveness of the commercialisation of this type of products under freezing, in terms of to the minimisation of the COPs formation. The cooking method (grilling) increased the COPs content in both types of shrimps, reaching 55.43 microg COPs/g fat in fresh shrimps and only 13.06 microg COPs/g fat in frozen ones.

Consequences of microwave heating and frying on the lipid fraction of chicken and beef patties.

Two types of commercial meat patties were analyzed to evaluate the effect of two applied cooking methods on the lipid fraction and the cholesterol oxidation process during heating. Microwave heating hardly modified the fatty acid profiles of both chicken and beef patties, whereas frying in olive oil increased oleic and eicosapentaenoic acids and decreased linoleic and docosahexaenoic acids in both types of products. Frying improved the omega6/omega3 fatty acids ratio in beef patties from 10.67 (raw) to 5.37 (fried). Total cholesterol oxidation product (COP) increments were 5.3-6.1-fold with microwave heating and 1.5-2.6-fold with frying. Chicken patties, raw and cooked, had a COP content twice as high as the corresponding beef ones.

Óxidos de colesterol: riesgos sanitarios y factores que condicionan su formación en alimentos / Cholesterol oxides: health risks and factors affecting their formation in food

Los óxidos de colesterol, responsables de ciertas actividades biológicas indeseables, parecen constituir un factor de riesgo en la incidencia de procesos ateroscleróticos. Estas posibles implicaciones negativas sobre la salud y su demostrada capacidad para ser absorbidos a través de la dieta han promovido el interés por la investigación de su presencia en alimentos y el estudio de la influencia de los factores que potencian su formación. Se ha podido constatar cómo el calentamiento de los alimentos, tanto durante el procesado tecnológico como en los tratamientos culinarios incrementan el contenido en óxidos de colesterol. La no disponibilidad de oxígeno y adecuadas condiciones de almacenamiento reducen la síntesis de estos compuestos (AU)

Oxidation process affecting fatty acids and cholesterol in fried and roasted salmon.

Salmon was processed by three different culinary techniques: pan-frying with olive oil, pan-frying with soya oil, and roasting. Roasting did not modify the fat content from that of raw samples. Frying increased the fat content 2-fold, with no difference between samples fried with different oils. Total cholesterol oxidized products (COPs) were 0.74, 2.98, 3.35, and 7.38 microg/g fat in raw, fried with olive oil, fried with soya oil, and roasted salmon, respectively, which represent 0.01, 0.08, 0.09, and 0.15% of cholesterol. A significant correlation (r = 0.902, < or = 0.01) was found between acidity index and total COPs. The most abundant COPs were 7-ketocholesterol, which appeared in all the samples, and cholestanetriol (one of the most citotoxic COP), which appeared only in cooked samples (1.05-1.33 microg/g fat). All cooked samples supplied more omega-6 polyunsaturated fatty acids (PUFAs) than raw samples and showed higher omega-6/omega-3 ratios. Roasted salmon showed the lowest omega-3 content and the highest PUFAs/(SFAs)-C18:0 and MUFAs+PUFAs/(SFAs-C18:0) ratios.

Fatty acid modifications and cholesterol oxidation in pork loin during frying at different temperatures.

The effect of frying with sunflower oil for 4 min at different temperatures (160, 170, and 180 degrees C) on fatty acids and cholesterol of pork loin meat was studied. Total fat content increased from 5.6% in fresh loin to 7.3, 7.8, and 12.1% at 160, 170, and 180 degrees C, respectively. Interactions with culinary fat gave rise to a significant increase in unsaturated acids/saturated acids and polyunsaturated acids/saturated acids ratios, which could be considered an advantage from a nutritional point of view. Less than 1 ppm (microg/g of sample) of cholesterol oxidation products was detected in fresh loin, whereas fried loin pork contained between 8.58 and 10.89 ppm. 7-Ketocholesterol (5.99 to 8.47 ppm in fried samples) and 7beta-hydroxycholesterol (1.43 to 2.55 ppm in fried samples) were the main cholesterol oxidation products. Cholestanetriol was not detected in any sample, and small quantities of 25-hydroxycholesterol and 5,6alpha-epoxycholesterol were found in the fried sample.

Quantitation of plasma membrane calcium pump phosphorylated intermediates by electrophoresis.

P-ATPases are characterized by the formation of acid-stable phosphorylated intermediates (EP) during their reaction cycle. We have developed a microscale method to determine EP that involves the phosphorylation of the enzyme using [gamma-(32)P]ATP and precipitation with TCA; separation of the sample by SDS-PAGE, and measurement of the enzyme protein and (32)P-labeled EP by digital analysis of both the stained gel and its autoradiogram, respectively. The principal advantages of this method over typical procedures (filtration and centrifugation) are the low amount of enzyme required and the substantial decrease in the blank values and data scattering produced by unspecific phosphorylation and nonquantitative recovering of the enzyme. Application of this new method to a purified preparation of the plasma membrane calcium ATPase (PMCA) results in overcoming the difficulties of measuring EP at high ATP concentrations. A biphasic behavior of the substrate curve for EP was observed when the study was extended to ATP levels within the physiological range. Since, in principle, the method does not require the use of highly purified preparations, it could be helpful for the study of phosphorylated intermediates especially under conditions in which small amounts of protein are available, e.g., mutated variants of P-ATPases.

Thermal stability of the plasma membrane calcium pump. Quantitative analysis of its dependence on lipid-protein interactions.

Thermal stability of plasma membrane Ca(2+) pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 +/- 12 kJ/mol in the range 33-45 degrees C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca(2+) pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K(LD)) showing that those phospholipids with higher affinity provide greater stability to the Ca(2+) pump. We developed a method for directly measure K(LD) by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K(LD) values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity.