Francesc González-Sastre

Plasma homocysteine is related to albumin excretion rate in patients with diabetes mellitus: a new link between diabetic nephropathy and cardiovascular disease?

Summary The high risk of cardiovascular disease in patients with diabetes mellitus, particularly in those with nephropathy, is not completely explained by classical risk factors. A high plasma homocysteine concentration is an independent risk factor for cardiovascular disease but information on its association with diabetes is limited. Fasting homocysteine concentrations were measured in the plasma of 165 diabetic patients (75 with insulin-dependent [IDDM]; 90 with non-insulin-dependent diabetes [NIDDM]) and 56 non-diabetic control subjects. Other measurements included the prevalence of diabetic complications, glycaemic control, lipid and lipoprotein levels, vitamin status and renal function tests. Patients with NIDDM had higher homocysteine levels than control subjects, whereas IDDM patients did not (9.2 ± 4.5 vs 7.7 ± 2 μmol/l, p < 0.01; and 7.0 ± 3 vs 7.4 ± 2 μmol/l, NS). Univariate correlations and multiple regression analysis showed albumin excretion rate to be the parameter with the strongest independent association with homocysteine. Patients with both types of diabetes and nephropathy had higher plasma homocysteine concentrations than those without nephropathy. Increases of homocysteine in plasma were related to increases in the severity of the nephropathy. Fasting hyperhomocysteinaemia was considered as the mean of the plasma homocysteine for all control subjects (7.5 ± 2.1 μmol/l) + 2 SD (cut-off =11.7 μmol/l). Nephropathy was present in 80 % of diabetic patients with fasting hyperhomocysteinaemia. In conclusion, increases in fasting homocysteine in diabetic patients are associated with increased albumin excretion rate, especially in those with NIDDM, thus providing a potential new link between microalbuminuria, diabetic nephropathy and cardiovascular disease. [Diabetologia (1998) 41: 684–693]

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.)

Increase of LDL susceptibility to oxidation occurring after intense, long duration aerobic exercise

The effect of heavy, long duration aerobic exercise on low density lipoprotein (LDL) susceptibility to oxidation and on distribution of LDL subfractions was studied. Six well-trained runners, previously fasted, ran continuously for 4 h. Controlled intake of liquid and food was permitted during exercise. Total plasma and LDL triglyceride increased significantly. LDL susceptibility to oxidation, measured as conjugated dienes formation, was modified significantly (P < or = 0.05) after running (14% reduction in lag phase time, and 8% increase in maximal curve slope). The percentage of electronegative LDL form (named LDLB) also increased significantly (P < or = 0.05) after exercise both basally (from 7.3% to 11%) and after 2h of induced oxidation (from 40.6% to 52.3%). Neither LDL susceptibility to oxidation nor increase of LDLB was statistically associated with food consumed during the race or modifications of triglycerides suggesting that this effect was due to exercise rather than food-related. The pattern of LDL subfractions was type A in all athletes before and after running. The oxidative LDL changes, seen in exercise conditions similar to those of hard training or competition, demonstrated an unfavourable effect of very intense exercise on lipoprotein metabolism.

LDL from aerobically-trained subjects shows higher resistance to oxidative modification than LDL from sedentary subjects

We studied the effect of regular intense aerobic exercise on the LDL susceptibility to oxidation and the electronegative LDL-proportion (LDL(-)). A group of 38 well-trained athletes was compared to a group of 38 age-BMI-matched sedentary individuals. Athletes showed higher concentration of total cholesterol (athletes 5.08 +/- 0.70 versus controls 4.65 +/- 0.75 mmol/l, P = 0.0229) and HDL cholesterol (athletes 1.72 +/- 0.47 versus controls 1.46 +/- 0.39 mmol/l, P = 0.0068); total plasma triglyceride, LDL cholesterol and VLDL cholesterol did not differ between trained and untrained subjects. The susceptibility of LDL to oxidation, determined by conjugated dienes formation and expressed as lag phase, was lower in athletes than in sedentaries (trained subjects 47.0 +/- 5.6 versus sedentary subjects 41.9 +/- 5.0 min, P = 0.0002). LDL(-) was similar in both groups (athletes 10.32 +/- 4.70 versus controls 10.26 +/- 3.71%). The antioxidant content in total plasma and isolated LDL (alpha-tocopherol, retinol, lycopene, alpha-carotene and beta-carotene) was quantitated by HPLC in a subgroup of 32 athletes and 32 control subjects. Athletes showed higher amounts of alpha-tocopherol and retinol in plasma, but not in LDL. However, none of these antioxidants correlated with the lag phase time. Trained subjects showed lower prevalence of smoking. However, no differences were observed between smokers and non-smokers concerning lag phase. No significant difference between athletes and sedentaries concerning LDL density, or composition was observed. We conclude that LDL from trained subjects is more resistant to oxidative modification than LDL from sedentary subjects. This observation could not be attributed to conventional antioxidants as alpha-tocopherol and carotene content of LDL was unchanged in trained subjects. Thus, although none of the variables studied appear as a single predictor of the LDL susceptibility to oxidation, an additive effect of the antioxidant content, the presence of some undetermined co-antioxidant, HDL and/or smoking habits cannot be discarded as responsible for the increased resistance to oxidation of LDL in trained subjects.

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.

Electronegative low density lipoprotein subform is increased in patients with short-duration IDDM and is closely related to glycaemic control

Summary We evaluated the effect of improving glycaemic control with intensive insulin therapy on LDL susceptibility to oxidation, electronegative LDL proportion, and LDL subfraction phenotype in a group of 25 patients with short-duration insulin-dependent diabetes mellitus (IDDM); 25 matched healthy control subjects were also studied. LDL susceptibility to oxidation was measured by continuous monitoring of conjugated diene formation. Electronegative LDL was isolated by anion exchange chromatography, and quantified as percentage of total LDL. Six LDL subfractions were isolated by density gradient ultracentrifugation and phenotype A or B classified as the quotient (LDL1-LDL3)/(LDL4-LDL6). Compared to the control group, IDDM subjects with poor glycaemic control showed higher electronegative LDL (19.03 ± 10.09 vs 9.59 ± 2.98 %, p < 0.001), similar LDL subfraction phenotype and lower susceptibility to oxidation (lag phase 45.6 ± 8.8 vs 41.2 ± 4.7 min, p < 0.05). After three months of intensive insulin therapy, HbA1 c decreased from 10.88 ± 2.43 to 5.69 ± 1.54 % (p < 0.001), and electronegative LDL to 13.84 ± 5.15 % (p < 0.05). No changes in LDL susceptibility to oxidation or LDL subfraction phenotype were observed. Electronegative LDL appeared significantly correlated to HbA1 c and fructosamine (p < 0.01 and p < 0.001) only in poorly controlled IDDM patients. These findings suggest that high electronegative LDL in IDDM subjects is related to the degree of glycaemic control, and could therefore be due to LDL glycation rather than to LDL oxidation or changes in LDL subfraction phenotype. [Diabetologia (1996) 39: 1469–1476]

Marathon runners presented lower serum cholesteryl ester transfer activity than sedentary subjects.

Acute exercise promotes raised HDL cholesterol concentrations by lipolysis stimulation, but this effect is insufficient to explain the more permanent HDL increases seen during regular exercise. During training periods in a group of marathon runners, we measured lipid transfer protein I (LTP-I)-mediated cholesteryl ester transfer activity (CETA) and its relationship to their HDL concentrations. Runners of both sexes showed significantly lower CETA values than those of sedentary controls. Male runners also had significantly lower serum concentrations of triglyceride, VLDL cholesterol and apolipoprotein B, and significantly higher concentrations of HDL cholesterol and apolipoprotein A-I than male controls. Results indicate that regular practice of aerobic exercise promotes modifications of lipoprotein metabolism related not only to lipolysis, but also to lower CETA. Such modifications are associated with reduced risk of atherosclerosis.

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

Susceptibility of plasma low- and high-density lipoproteins to oxidation in patients with severe hyperhomocysteinemia

Abstract A moderate increase in plasma homocysteine is increasingly considered an important risk factor of atherosclerosis and thrombosis. However, the mechanisms by which hyperhomocysteinemia induces vascular disease are not well defined. In vitro studies suggest that cysteine and homocysteine can induce oxidative modification of low-density lipoproteins (LDL). This suggestion is relevant because lipoprotein oxidation is thought to play a key role in the development of atherosclerosis and in the triggering of thrombotic events. An attractive model to study this topic is provided by patients with classical homocystinuria, an inherited disease characterized by severe hyperhomocysteinemia and a high incidence of thromboembolisms. We investigated the existence of oxidized LDL and the susceptibility to oxidation of the plasma cholesterol-rich lipoproteins in six patients with severe hyperhomocysteinemia, most likely due to classical homocystinuria, and compared the results with matched controls. The proportion of electronegative LDL and the concentration of thiobarbituric acid reactive substances in native LDL and high-density lipoproteins (HDL) did not differ between patients and controls, suggesting that the proportion of modified lipoproteins is not increased in patients with severe hyperhomocysteinemia. The susceptibility to oxidative modification of plasma LDL and HDL was also similar in the two groups, although the patients had homocysteine levels 18.3-fold higher than controls. Thus, increased oxidative modification is not likely to be a relevant mechanism in explaining their high incidence of vascular disease. A possible explanation for the lack of increased susceptibility to oxidation, as would be expected for the metabolic blockade that causes classical homocystinuria, is the 4.1-fold decrease in the concentration of cysteine in the plasma of patients. As a result the total concentration of homocysteine plus cysteine was slighty lower in patients than in controls. This interpretation implies that more studies are needed on lipoprotein susceptibility to oxidation in patients in which both plasma homocysteine and cysteine concentrations are increased. This metabolic situation may be frequent in the population with moderate hyperhomocysteinemia and vascular disease.

Increased production of very-low-density lipoproteins in transgenic mice overexpressing human apolipoprotein A-II and fed with a high-fat diet.

We investigated the mechanisms that lead to combined hyperlipidemia in transgenic mice that overexpress human apolipoprotein (apo) A-II (line 11.1). The 11.1 transgenic mice develop pronounced hypertriglyceridemia, and a moderate increase in free fatty acid (FFA) and plasma cholesterol, especially when fed a high-fat/high-cholesterol diet. Post-heparin plasma lipoprotein lipase and hepatic lipase activities (using artificial or natural autologous substrates), the decay of plasma triglycerides with fasting, and the fractional catabolic rate of the radiolabeled VLDL-triglyceride (both fasting and postprandial) were similar in 11. 1 transgenic mice and in control mice. In contrast, a 2.5-fold increase in hepatic VLDL-triglyceride production was observed in 11. 1 transgenic mice in a period of 2 h in which blood lipolysis was inhibited. This increased synthesis of hepatic VLDL-triglyceride used preformed FFA rather than FFA of de novo hepatic synthesis. The 11.1 transgenic mice also presented reduced epididymal/parametrial white adipose tissue weight (1.5-fold), increased rate of epididymal/parametrial hormone-sensitive lipase-mediated lipolysis (1.2-fold) and an increase in cholesterol and, especially, in triglyceride liver content, suggesting an enhanced mobilization of fat as the source of preformed FFA reaching the liver. Increased plasma FFA was reverted by insulin, demonstrating that 11.1 transgenic mice are not insulin resistant. We conclude that the overexpression of human apoA-II in transgenic mice induces combined hyperlipidemia through an increase in VLDL production. These mice will be useful in the study of molecular mechanisms that regulate the overproduction of VLDL, a situation of major pathophysiological interest since it is the basic mechanism underlying familial combined hyperlipidemia.