Oscar Jorba

Platelet-Activating Factor Acetylhydrolase Is Mainly Associated With Electronegative Low-Density Lipoprotein Subfraction

Background Electronegative LDL [LDL(−)], a modified subfraction of LDL present in plasma, induces the release of interleukin‐8 and monocyte chemotactic protein‐1 from cultured endothelial cells. Methods and Results We demonstrate that platelet‐activating factor acetylhydrolase (PAF‐AH) is mainly associated with LDL(−). LDL(−) had 5‐fold higher PAF‐AH activity than the nonelectronegative LDL subfraction [LDL(+)] in both normolipemic and familial hypercholesterolemic subjects. Western blot analysis after SDS‐PAGE confirmed these results, because a single band of 44 kDa corresponding to PAF‐AH appeared in LDL(−) but not in LDL(+). Nondenaturing polyacrylamide gradient gel electrophoresis demonstrated that PAF‐AH was bound to LDL(−) regardless of LDL size. In accordance with the above findings, nonesterified fatty acids, a cleavage product of PAF‐AH, were increased in LDL(−) compared with LDL(+). Conclusions The high PAF‐AH activity observed in LDL(−) could be related to the proinflammatory activity of these lipoproteins toward cultured endothelial cells. (Circulation. 2003;108:92‐96.)

Effect of Simvastatin Treatment on the Electronegative Low-Density Lipoprotein Present in Patients With Heterozygous Familial Hypercholesterolemia

Most described modifications of low-density lipoprotein (LDL) cholesterol share an increase in its negative electric charge; in fact, an electronegative form of LDL can be identified and isolated from plasma. Although the exact nature of the chemical modification of electronegative LDL is still controversial, its toxicity on endothelial cells has been demonstrated. Statins have protective effects against cardiovascular disease that are independent of their lipid-lowering action and which could be due, at least in part, to the prevention of LDL modification. We evaluated the effect of 6 months of simvastatin therapy (40 mg/day) on electronegative LDL proportion and LDL susceptibility to in vitro induced oxidation in 21 patients with heterozygous familial hypercholesterolemia (FH). Eleven normolipemic subjects were analyzed as a control group. Total cholesterol as well as LDL and very low density lipoprotein cholesterol, triglycerides, and apoprotein B decreased 30% after the first month of therapy, with no further decreases thereafter. LDL susceptibility to oxidation was similar in FH patients and controls and did not change throughout the treatment. Electronegative LDL proportion was 35.1 +/- 9.9% in FH patients and 9.1 +/- 2.4% in control subjects (p <0.0001) but, in contrast to total LDL cholesterol and the rest of lipid parameters, it decreased to 28.6 +/- 9.1% in the third month and to 21.2 +/- 7.7% in the sixth month of therapy. The decrease in these cytotoxic particles may be a relevant mechanism by which simvastatin protects against cardiovascular disease.

Effect of physical exercise on lipoprotein(a) and low-density lipoprotein modifications in type 1 and type 2 diabetic patients

To evaluate the effect of physical exercise on blood pressure, the lipid profile, lipoprotein(a) (Lp(a)), and low-density lipoprotein (LDL) modifications in untrained diabetics, 27 diabetic patients (14 type 1 and 13 type 2) under acceptable and stable glycemic control were studied before and after a supervised 3-month physical exercise program. Anthropometric parameters, insulin requirements, blood pressure, the lipid profile, Lp(a), LDL composition, size, and susceptibility to oxidation, and the proportion of electronegative LDL (LDL(-)) were measured. After 3 months of physical exercise, physical fitness improved (maximal O2 consumption [VO2max], 29.6 +/- 6.8 v 33.0 +/- 8.4 mL/kg/min, P < .01). The body mass index (BMI) did not change, but the waist circumference (83.2 +/- 11.8 to 81.4 +/- 11.2 cm, P < .05) decreased significantly. An increase in the subscapular to triceps skinfold ratio (0.91 +/- 0.37 v 1.12 +/- 0.47 cm, P < .01) and midarm muscle circumference ([MMC], 23.1 +/- 3.4 v 24.4 +/- 3.7 cm, P < .001) were observed after exercise. Insulin requirements (0.40 +/- 0.18 v 0.31 +/- 0.19 U/kg/d, P < .05) and diastolic blood pressure (80.2 +/- 10 v 73.8 +/- 5 mm Hg, P < .01) decreased in type 2 diabetic patients. High-density lipoprotein cholesterol (HDL-C) increased in type 1 patients (1.48 +/- 0.45 v1.66 +/- 0.6 mmol/L, P < .05), while LDL cholesterol (LDL-C) decreased in type 2 patients (3.6 +/- 1.0 v3.4 +/- 0.9 mmol/L, P < .01). Although Lp(a) levels did not vary in the whole group, a significant decrease was noted in patients with baseline Lp(a) above 300 mg/L (mean decrease, -13%). A relationship between baseline Lp(a) and the change in Lp(a) (r = -.718, P < .0001) was also observed. After the exercise program, 3 of 4 patients with LDL phenotype B changed to LDL phenotype A, and the proportion of LDL(-) tended to decrease (16.5% +/- 7.4% v 14.0% +/- 5.1%, P = .06). No changes were observed for LDL composition or susceptibility to oxidation. In addition to its known beneficial effects on the classic cardiovascular risk factors, regular physical exercise may reduce the risk of cardiovascular disease in diabetic patients by reducing Lp(a) levels in those with elevated Lp(a) and producing favorable qualitative LDL modifications.

Novel phospholipolytic activities associated with electronegative low-density lipoprotein are involved in increased self-aggregation.

Electronegative low-density lipoprotein (LDL(-)) is a minor LDL subfraction present in plasma with increased platelet-activating factor acetylhydrolase (PAF-AH) activity. This activity could be involved in the proinflammatory effects of LDL(-). Our aim was to study the presence of additional phospholipolytic activities in LDL(-). Total LDL was fractionated into electropositive (LDL(+)) and LDL(-) by anion-exchange chromatography, and phospholipolytic activities were measured by fluorometric methods. Phospholipolytic activity was absent in LDL(+) whereas LDL(-) presented activity against lysophosphatidylcholine (LPC, 82.4 +/- 34.9 milliunits/mg of apoB), sphingomyelin (SM, 53.3 +/- 22.5 milliunits/mg of apoB), and phosphatidylcholine (PC, 25.7 +/- 4.3 milliunits/mg of apoB). LDL(-), but not LDL(+), presented spontaneous self-aggregation at 37 degrees C in parallel to phospholipid degradation. This was observed in the absence of lipid peroxidation and suggests the involvement of phospholipolytic activity in self-aggregation of LDL(-). Phospholipolytic activity was not due to PAF-AH, apoE, or apoC-III and was not increased in LDL(+) modified by Cu (2+) oxidation, acetylation, or secretory phospholipase A 2 (PLA 2). However, LDL(-) efficiently degraded phospholipids of lipoproteins enriched in LPC, such as oxidized LDL or PLA 2-LDL, but not native or acetylated LDL. This finding supports that LPC is the best substrate for LDL(-)-associated phospholipolytic activity. These results reveal novel properties of LDL(-) that could play a significant role in its atherogenic properties.

Ascorbic acid inhibits the increase in low-density lipoprotein (LDL) susceptibility to oxidation and the proportion of electronegative LDL induced by intense aerobic exercise.

BackgroundWe have previously reported the finding of an acute increment in the susceptibility of low-density lipoprotein (LDL) to oxidation and in the proportion of electronegative LDL [LDL(−)] after intense exercise. We have now studied the effect of oral supplementation with 1 g ascorbic acid, immediately before a 4-h athletic race, on the susceptibility of LDL to oxidation, the proportion of LDL(−), and the α-tocopherol and lipid peroxides content in LDL, in order to inhibit such deleterious changes, and to confirm the oxidative nature of modifications of LDL induced by exercise. MethodsWe studied seven highly trained runners who received a supplement of 1 g ascorbic acid and a control group of seven who did not receive the supplement. The susceptibility of LDL to oxidation was assessed by measurement of conjugated dienes after CuSO4-induced oxidation, the proportion of LDL(−) was determined by anion exchange chromatography, α-tocopherol was quantified by reverse-phase high performance liquid chromatography, and lipid peroxides were measured by the thiobarbituric acid-reactive substances (TBARS) method. ResultsAfter exercise, in the control group there was an increase in both the susceptibility of LDL to oxidation (change in lag phase from 51.4 ± 4.7 min to 47.0 ± 4.6 min, P < 0.05) and the proportion of LDL(−) (from 11.1 ± 1.4% to 13.0 ± 2.2%, P<0.05), but these did not occur in the ascorbic acid group (change in lag phase from 49.7 ± 2.3 min to 50.4 ± 4.2 min, and in LDL(−) from 9.7 ± 1.7% to 10.1 ± 1.7%). No significant changes in the absolute amount of LDL α-tocopherol were observed after exercise (ascorbic acid group: 6.65 ± 0.94 mol/mol apoB before the race, 7.13 ± 0.88 mol/mol apoB after the race; control group: 7.34 ± 0.69 mol/mol apoB before the race, 7.06 ± 0.69 mol/mol apoB after the race), but significant differences were found when increments or decrements of α-tocopherol were tested (α-tocopherol increased 9.9 ± 11.5% in the ascorbic acid group, and decreased 0.6 ± 7.3% in the control group; P<0.018). TBARS did not change after exercise. ConclusionsWe conclude that 1 g ascorbic acid inhibits the increase in LDL susceptibility to oxidation after exercise, preventing this acute pro-atherogenic effect. In addition, the observation that LDL(−) enhancement is prevented by ascorbic acid supports the hypothesis that at least some of the circulating LDL(−) originates from oxidative processes. Coronary Artery Dis 9:249–255

Electronegative low-density lipoprotein subfraction from type 2 diabetic subjects is proatherogenic and unrelated to glycemic control

The physicochemical and biological characteristics of electronegative low‐density lipoprotein (LDL) (LDL(−)) from type 2 diabetic patients (DM2), before and after insulin therapy, were studied.

Effect of improving glycemic control on low-density lipoprotein particle size in type 2 diabetes

The current study sought to assess the effect of improving glycemic control in type 2 diabetes on the components of diabetic dyslipidemia, especially low-density lipoprotein (LDL) size. A total of 33 type 2 diabetic patients (48.5% women, age 59.6 +/- 11.1 years, body mass index [BMI] 28.9 +/- 4.9, diabetes duration 6 [0 to 40] years, 40.7% on insulin) were seen at the hospital because of poor glycemic control (hemoglobin A(1c) [HbA(1c)] 10.33% +/- 1.89%). Triglyceride, LDL-cholesterol (LDLc, Friedewald/ ultracentrifugation), high-density lipoprotein HDL-cholesterol (HDLc, direct method), apolipoproteins AI (apoAI) and B (apoB) (immunoturbidimetry), and LDL size (gradient gel electrophoresis) were measured at baseline and after improvement in glycemic control (decrease >/= 1 percentage point in HbA(1c) and final HbA(1c) </= 8%). Improvement in glycemic control (HbA(1c) 7.01% +/- 0.63%, P <.0005 v baseline) after a follow-up of 3.5 (range, 1 to 13) months resulted in a significant reduction in LDLc (3.34 +/- 1.02 v 3.62 +/- 1.15 mmol/L, P <.05) and apoB (1.07 +/- 0.25 v 1.17 +/- 0.29 g/L, P <.01) and an increase in HDLc (1.21 +/- 0.32 v 1.13 +/- 0.34 mmol/L, P <.05) and apoAI (1.36 +/- 0.24 v 1.27 +/- 0.24 mmol/L, P < 0.01) in the whole group, and an increase in LDL particle size (25.61 +/- 0.53 v 25.10 +/- 0.31 nm, P <.005) in the 14 patients showing LDL phenotype B at baseline. No significant changes were seen in body weight or BMI. We conclude that improvement of glycemic control in type 2 diabetes improves most of the components of diabetic dyslipidemia, including a shift towards larger LDL particles in subjects with phenotype B.

Postprandial lipidemia is normal in non-obese type 2 diabetic patients with relatively preserved insulin secretion.

To assess postprandial lipidemia in normotriglyceridaemic type 2 diabetic patients treated with diet only, 12 non-obese patients (8 males, hemoglobin A(1c) [HbA(1c)] 6.80 +/- 0.67%) and 14 controls of similar age, body mass index (BMI), and fasting triglyceride (Tg) were given a test meal (58 g fat, 100,000 IU vitamin A). Fasting low-density lipoprotein (LDL) cholesterol (LDLc), high-density lipoprotein (HDL) cholesterol (HDLc), free fatty acids, and apolipoprotein B (apoB), and fasting and postprandial Tg, retinylpalmitate (RP), LDL size, glucose, and insulin were measured. The homeostasis assessment model (HOMA) index and lipoprotein (Lpl) and hepatic (HL) lipase activities were estimated. Patients showed lower fasting HDLc (1.12 +/- 0.26 v 1.40 +/- 0.28 mmol/L, P =.02) and a trend towards smaller LDL particles, which was significant 4 hours postprandially (25.86 +/- 0.40 v 26.16 +/- 0.30 nm, P =.04). The area under the curve of Tg (AUC-Tg) and RP, and Lpl were similar, but HL was higher in patients (156.63 +/- 23.89 v 118 +/- 43.27 U/L, P =.011). HL correlated inversely with LDL size and directly with the HOMA index. In conclusion, normotriglyceridemic type 2 diabetic patients with insulin resistance but relatively preserved insulin secretion show low fasting HDLc and increased HL, but normal postprandial lipidemia.