Javier S. Perona

Alteration of Lipids, G Proteins, and PKC in Cell Membranes of Elderly Hypertensives

Abstract—In this study, we quantified the levels of lipids and signaling proteins in erythrocyte membranes from elderly normotensive and hypertensive subjects. In hypertensive subjects, the cholesterol/phospholipid ratio increased significantly in erythrocyte membranes, owing to the reduction of phospholipid levels concomitant with a rise in the levels of cholesterol. In addition, differences were also found in the amount of fatty acids in both phospholipid and cholesterol esters. Erythrocyte membranes from hypertensive subjects contained higher levels of monounsaturated and lower levels of polyunsaturated fatty acids. On the other hand, signaling proteins such as G proteins and protein kinase C have been implicated in the control of blood pressure. Previous studies have shown that the cellular localization and the activity of these proteins are modulated by the type and the abundance of membrane lipids. For this reason, we assessed the levels of these signaling molecules in the membrane. We found that the levels of membrane-associated (active/preactive) G proteins (G&agr;i, G&agr;o, and G&bgr;) and protein kinase C were significantly reduced in hypertensive subjects. We believe that these alterations could be related to the etiopathology of hypertension in elderly subjects or alternatively may correspond to adaptive compensatory mechanisms.

Fatty acid composition of chylomicron remnant-like particles influences their uptake and induction of lipid accumulation in macrophages

The influence of the fatty acid composition of chylomicron remnant‐like particles (CRLPs) on their uptake and induction of lipid accumulation in macrophages was studied. CRLPs containing triacylglycerol enriched in saturated, monounsaturated, n−6 or n−3 polyunsaturated fatty acids derived from palm, olive, corn or fish oil, respectively, and macrophages derived from the human monocyte cell line THP‐1 were used. Lipid accumulation (triacylglycerol and cholesterol) in the cells was measured after incubation with CRLPs for 5, 24 and 48 h, and uptake over 24 h was determined using CRLPs radiolabelled with [3H]triolein. Total lipid accumulation in the macrophages was significantly greater with palm CRLPs than with the other three types of particle. This was mainly due to increased triacylglycerol concentrations, whereas changes in cholesterol concentrations did not reach significance. There were no significant differences in lipid accumulation after incubation with olive, corn or fish CRLPs. Palm and olive CRLPs were taken up by the cells at a similar rate, which was considerably faster than that observed with corn and fish CRLPs. These findings demonstrate that CRLPs enriched in saturated or monounsaturated fatty acids are taken up more rapidly by macrophages than those enriched in n−6 or n−3 polunsaturated fatty acids, and that the faster uptake rate results in greater lipid accumulation in the case of saturated fatty acid‐rich particles, but not monounsaturated fatty acid‐rich particles. Thus, dietary saturated fatty acids carried in chylomicron remnants may enhance their propensity to induce macrophage foam cell formation.

Long-chain fatty alcohols from pomace olive oil modulate the release of proinflammatory mediators

Pomace olive oil is a by-product of olive oil extraction that is traditionally produced and consumed in Spain. The nonglyceride matter of this oil is a good source of interesting minor compounds, like long-chain fatty alcohols, which are present free or as part of waxes. In the present study, long-chain fatty alcohols were isolated from the nonglyceride fraction of pomace olive oil, and the composition was identified and quantified. The major components of long-chain fatty alcohols were tetracosanol, hexacosanol and octacosanol. We investigated the ability of long-chain fatty alcohols from pomace olive oil to inhibit the release of different proinflammatory mediators in vitro by cells involved in inflammatory processes. Long-chain fatty alcohols significantly and dose-dependently decreased nitric oxide production by RAW 264.7 murine macrophages stimulated with lipopolysaccharide. Western blot analysis showed that nitric oxide reduction was a consequence of the inhibition of inducible nitric oxide synthetase expression. Long-chain fatty alcohols also reduced tumor necrosis factor-alpha and prostaglandin E(2) production, although the potency of inhibition for the latter was lower. On the other hand, long-chain fatty alcohols significantly reduced thromboxane A(2) production in rat peritoneal neutrophils stimulated with the calcium ionophore A-23187. The reduction of eicosanoid release was related to the inhibition of phospholipase A(2) enzyme activity by long-chain fatty alcohols, reaching an inhibitory concentration 50% value of 6.2 microg/ml. These results showed that long-chain fatty alcohols may have a protective effect on some mediators involved in the inflammatory damage development, suggesting its potential value as a putative functional component of pomace olive oil.

Mediterranean-Style Diet Effect on the Structural Properties of the Erythrocyte Cell Membrane of Hypertensive Patients: The Prevencion con Dieta Mediterranea Study

A currently ongoing randomized trial has revealed that the Mediterranean diet, rich in virgin olive oil or nuts, reduces systolic blood pressure in high-risk cardiovascular patients. Here, we present a structural substudy to assess the effect of a Mediterranean-style diet supplemented with nuts or virgin olive oil on erythrocyte membrane properties in 36 hypertensive participants after 1 year of intervention. Erythrocyte membrane lipid composition, structural properties of reconstituted erythrocyte membranes, and serum concentrations of inflammatory markers are reported. After the intervention, the membrane cholesterol content decreased, whereas that of phospholipids increased in all of the dietary groups; the diminishing cholesterol:phospholipid ratio could be associated with an increase in the membrane fluidity. Moreover, reconstituted membranes from the nuts and virgin olive oil groups showed a higher propensity to form a nonlamellar inverted hexagonal phase structure that was related to an increase in phosphatidylethanolamine lipid class. These data suggest that the Mediterranean-style diet affects the lipid metabolism that is altered in hypertensive patients, influencing the structural membrane properties. The erythrocyte membrane modulation described provides insight in the structural bases underlying the beneficial effect of a Mediterranean-style diet in hypertensive subjects.

Simultaneous determination of molecular species of monoacylglycerols, diacylglycerols and triacylglycerols in human very-low-density lipoproteins by reversed-phase liquid chromatography

The aim of the present study was to investigate the applicability of a previously developed method for the analysis of triacylglycerol molecular species to the simultaneous determination of triacylglycerols, diacylglycerols and monoacylglycerols of human very-low-density lipoproteins (VLDL). Ten elderly women were recruited for the study. Blood was obtained in fasting conditions and VLDL were isolated by ultracentrifugation. Neutral lipids were separated by solid-phase extraction and were subsequently injected on a reversed-phase HPLC system, with an elution system composed of acetone in acetonitrile. The method allowed the separation of four monoacylglycerols, 18 diacylglycerols and 24 triacylglycerols, including the resolution of positional isomers of diacylglycerols. Monoacylglycerols were composed of oleic, linoleic, palmitic and stearic acids. The major diacylglycerols were 1,2-dilinoleoyl-glycerol and 1,3-dilinoleoyl-glycerol (14.24+/-1.02 and 17.93+/-1.42%, respectively). The main triacylglycerols quantified were dioleoyl-stearoyl-glycerol (OOS), oleoyl-dipalmitoyl-glycerol (OPP), trilinoleoyl-glycerol (LLL) and linoleoyl-distearoyl-glycerol (LSS), accounting for 11.25+/-2.15, 10.14+/-2.05, 9.35+/-2.30 and 8.56+/-1.56%, respectively. An inverse relationship between polarity and fatty acid disappearance from triacylglycerols (r(2)=0.82, P<0.05) and from diacylglycerols (r(2)=0.93, P<0.01) was discovered. In conclusion, the method allowed, for the first time, the easy, rapid and simultaneous determination in a single chromatogram of triacylglycerol, diacylglycerol and monoacylglycerol molecular species of human VLDL by reversed-phase HPLC.

CHARACTERIZATION OF THE TRIACYLGLYCEROL MOLECULAR SPECIES OF FISH OIL BY REVERSED-PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

The nutritional benefits attributed to fish oils have been the basis for the study of the structural composition of Sardine oil triacylglycerols (TAGs). TAGs were separated reversed-phase high performance liquid chromatography (RP-HPLC) with the result of 65 chromatographic peaks resolved. The problem of identification was avoided by the use of two more chromatographic techniques. Separation of the sardine oil TAGs into fractions by silver-ion thin layer chromatography (TLC) and its subsequent fatty acid analysis by gas chromatography allowed identification of 59 of the 65 chromatographic peaks. From those peaks, the major was trimyristin (MMM), with 8.22 % of the total. Dioleoyl-acyl-glycerol (OPO, OOE), dipalmitoyl-acyl-glycerol (PPO, PPPo), dipalmitoleoyl-acyl.glycerol (PoPoO) and dieicosapentaenoyl-acyl-glycerol (EEP) species were found in important amounts.

Influence of different dietary fats on triacylglycerol deposition in rat adipose tissue

It has been demonstrated that triacylglycerol (TAG) mobilization from adipose tissue is selective and depends on fatty acid (FA) chain length, unsaturation and positional isomerism. The present study was performed to determine the influence of dietary fat on the composition of TAG stored in rat perirenal and subcutaneous adipose tissues. These results may provide information on the susceptibility of stored TAG to hydrolysis and further mobilization, and may help to establish an interrelationship between dietary composition and the FA efflux from adipose tissue. TAG molecular species and FA composition were determined by HPLC and GLC respectively. No significant differences were found in either FA or TAG composition between perirenal and subcutaneous adipose depots. The major FA in the dietary fats were present in the adipose tissues of the animals; in most cases, in similar proportions. However, differences were found between dietary and adipose tissue content of minor FA, which suggests that dietary FA composition is altered between ingestion and deposition in adipose tissue. The TAG molecular species of rat adipose tissue were enriched with the FA characteristic of each dietary fat. Dietary sunflower oil was responsible for enrichment with the most polar TAG. This finding may suggest easier mobilization of stored TAG. In conclusion, the process of fatty acid and TAG deposition in rat adipose tissue is selective, and depends on the composition of the diet.

Evaluation of the Effect of Dietary Virgin Olive Oil on Blood Pressure and Lipid Composition of Serum and Low-Density Lipoprotein in Elderly Type 2 Diabetic Subjects

Dietary virgin olive oil may help to reduce blood pressure in hypertensive individuals, but little is known about the effect on type 2 diabetic patients. For the present study, 17 type 2 diabetic elderly subjects and 23 healthy elderly controls received a diet rich in virgin olive oil for 4 weeks. Blood pressure, biochemical parameters, low-density lipoprotein (LDL), and oxidized LDL lipids and fatty acids were measured. Systolic blood pressure was reduced after virgin olive oil consumption in both controls and diabetic patients. Although the biochemical parameters were not modified, the intervention protected LDL from oxidation and restored the levels of dihomo-gamma-linolenic acid (20:3, n-6) in serum cholesterol esters and phospholipids of diabetic patients. In conclusion, the present study provides new evidence of the effects of dietary virgin olive oil on blood pressure and LDL oxidation in type 2 diabetics. It is likely that the components responsible for the observed effects are the monounsaturated fatty acids and the presence of antioxidants in the oil, but this needs further investigation.

Plasma lipid modifications in elderly people after administration of two virgin olive oils of the same variety (Olea europaea var. hojiblanca) with different triacylglycerol composition

In the present study we examined whether two virgin olive oils (VOO1 and VOO2), of the same variety (Olea europaea var. hojiblanca with a similar composition of minor components but differing in the content of triacylglycerol molecular species, had different effects on blood pressure and plasma lipid levels in a healthy elderly population. Thirty-one participants, aged 84-9 (SD 6.4) years, were asked to participate in the study. No differences were found with regard to blood pressure after both experimental periods (VOO1 and VOO2). However, plasma total cholesterol and LDL-cholesterol were reduced only after VOO1 (P<0.01). The reduction of plasma cholesterol concentrations was related to the incorporation of oleic acid into plasma cholesteryl esters and phospholipids strongly correlated with plasma total cholesterol and LDL-cholesterol levels in all experimental periods studied (r2>0.418, P<0.07), except for phospholipids in VOO1 (P=0.130 for total cholesterol and p=0.360 for LDL-cholesterol). These results have demonstrated that blood pressure and plasma lipids can be modified by the consumption of VOO in elderly people, but that the extent of such modification depends on the composition and amount of active minor components and triacylglycerol molecular species.

Determination of rat liver triglycerides by gas–liquid chromatography and reversed-phase high-performance liquid chromatography

Rats fed with a fat-free or an olive oil-rich diet were employed to compare the response of two chromatographic techniques in the determination of rat liver triglyceride (TG) molecular species composition. Gas-liquid chromatography (GLC) on polarizable liquid phase and reversed-phase high-performance liquid chromatography (RP-HPLC) have been commonly employed for TG analysis, obtaining a similar number of chromatographic peaks when used for animal tissue TG determination. In the present study similar results were achieved with regard to most relevant chromatographic peaks, however, important differences were found in the content of minor TGs. Indeed, RP-HPLC permitted separation of long chain polyunsaturated fatty acids, which were not detected by GLC, while the latter technique reported a higher number of myristoyl-containing TG species. RP-HPLC analysis reported a greater number of TGs, with more similarity to a random composition, made up from the liver fatty acid composition. Therefore, it was concluded that utilization of both techniques would be helpful for liver TG analysis as the use of only one of them does not provide a complete profile of liver TGs. Nevertheless RP-HPLC seems to be more useful for this purpose since revealed a more extensive profile.