Silvia Sanguinetti

Low-density lipoprotein composition and oxidability in atherosclerotic cardiovascular disease.

OBJECTIVES To characterize low-density lipoprotein (LDL) chemical composition and oxidability in normolipidemic and dyslipidemic patients with atherosclerotic cardiovascular disease, as compared with matched control subjects. To evaluate LDL susceptibility to oxidation, we determined the cutoff points of thiobarbituric reactive substances (TBARS) in LDL after oxidative stress, as well as its resistance to oxidation. DESIGN AND METHODS LDL (density 1.019-1.063 g/mL) of 24 men with atherosclerotic cardiovascular disease (12 normolipidemic and 12 dyslipidemic patients) and 18 age-matched healthy control men. LDL chemical composition was determined and apo B/cholesterol ratio was calculated. TBARS in native LDL and after 60 and 120 min of LDL oxidation with copper were measured. The conjugated diene production kinetics during LDL incubation with copper were also studied, lag time being an oxidation resistance marker. Cutoff points for the positivity criterion of apoB/cholesterol ratio in LDL and TBARS in native and oxidized LDL were evaluated using the receiver operator characteristic (ROC) graphic method. RESULTS LDL were triglyceride-enriched, the apoB/cholesterol ratio being higher in patients than in controls, without differences between normolipidemic and dyslipidemic subgroups. We have established the following cutoff values to differentiate between patients and controls: 0.43 mg/mg for the apo B/cholesterol ratio in LDL; 3.0 nmol malondialdehyde/mg protein for TBARS in native LDL; 22 and 80 nmol malondialdehyde/mg protein after 60- and 120-min postoxidative stress, respectively. We did not find differences in the conjugated diene production kinetics between patients and controls. CONCLUSIONS The enrichment in triglycerides and the high apoB/ cholesterol ratio suggest the presence of an abnormal LDL particle in normolipidemic and dyslipidemic patients. This LDL particle was more susceptible to oxidation. In the ROC analysis, the TBARS plot at 120 min exhibited greater accuracy and better performance than the other LDL oxidability markers.

Hypercholesterolemia attenuates postischemic ventricular dysfunction in the isolated rabbit heart

The effects of the chronic administration of cholesterol on the stunned myocardium have not been studied. The objective was to determine the effect of a cholesterol enriched diet on postischemic ventricular dysfunction. In group 1 (G1, n = 7 isolated rabbit hearts underwent a follow up of ventricular function during 30 min in aerobic conditions. In group 2 (G2, n = 6) G1 was repeated but the animals were subjected to a 1% cholesterol enriched diet during 4 weeks (hypercholesterolemic animals). In group 3 (G3, n = 8) hearts underwent 15 min of global ischemia followed by 30 min of reperfusion. In Group 4 (G4, n = 11) G3 was repeated, but in hypercholesterolemic animals. Since cholesterol decreased the inotropism in basal situation, and this makes the comparison between groups difficult, we performed a Group 5 (G5, n = 7), in which G4 protocol was repeated but isoproterenol (8 μg/kg/min) was administered 10 min before ischemia, in order to match the preischemic inotropic state with respect to the normocholesterolemic ones. G1 and G2 maintained a stable inotropism during the 30 min of perfusion. The preischemic left ventricular developed pressure (LVDP) in G3 and G4 was 91.4± 4.3 and 70.8± 3.4 mmHg (p< 0.05), respectively, and after 30 min of reperfusion differences were not observed between G3 and G4. Nevertheless, when LVDP is expressed as a percentage, we detected an attenuation of postischemic systolic alterations in hypercholesterolemic animals (67.3± 3.6 in G4 vs. 90.8± 3.1% in G3, p< 0.05). When LVDP in G5 was increased until matching the one of G3, there were no differences after 30 min of reperfusion. Left ventricular end diastolic pressure increased 285± 46%, 61± 25% (p< 0.05 vs. G3 and G5) and 216± 25% in G3, G4 and G5 at 30 min of reperfusion. There were no differences either in the values of tau or infarct size between groups. Thus, in hypercholesterolemic animals, a decrease of the preischemic inotropism exists and there is an attenuation of the stunned myocardium. When contractility of the normo and hypercholesterolemic animals is matched, the beneficial effect disappears.

Detection of structural alterations in LDL isolated from type 2 diabetic patients: application of the fructosamine assay to evaluate the extent of LDL glycation

Modifications in LDL such as glycation contribute to the accelerated development of macrovascular alterations in diabetic patients [1], but to date there is no recommended method to determine the extent of plasma LDL glycation. The fructosamine assay originally described by Johnson et al. [2] to evaluate medium term glycemic control in diabetic patients, was previously applied to measurements of glycated protein in LDL fractions isolated from in vitro glycated plasma obtained from non-diabetic subjects [3]. However, fructosamine values in LDL from diabetic patients are as yet unknown. Therefore, we re-adapted and evaluated the fructosamine assay for the determination of glycated LDL in type 2 diabetic patients. Besides, we characterized LDLs by means of their chemical composition and estimated the predominance of small dense LDL through the total proteins/cholesterol ratio in the LDL fraction [4]. Twenty-three type 2 diabetic patients of either sex, whose ages ranged from 47 to 82 years, were studied. Mean (9S.D.) levels of HbA1c were 8.592.5%, serum fructosamine 370990 mmol/l and glycemia in the fasting state 213956 mg/dl. Mean (9S.D.) levels of plasma triglycerides (TG) and total, HDLand LDLcholesterol were 173976, 227942, 49913 and 1419 36 mg/dl, respectively. Throughout, there was no clinical or laboratory evidence of impaired liver or kidney function. The control group comprised 19 subjects of either sex, ages ranging from 22 to 87 years, whose mean (9S.D.) levels of HbA1c were 4.990.5%, serum fructosamine 230910 mmol/l and glycemia in the fasting state 8697 mg/dl. Mean (9S.D.) levels of plasma triglycerides and total, HDLand LDLcholesterol were 85938, 197944, 59916 and 118943 mg/dl, respectively. LDL was isolated from fasting plasma supplemented with EDTA 1 g/l, by sequential ultracentrifugation within the 1.019–1.063 g/ml density range. The proposed assay was evaluated as below. An LDL aliquot obtained from controls was used to make up a pool to check the correlation between the degree of in vitro glycation and the measurement of fructosamine in the LDL fraction isolated, as well as to determine assay recovery and reproducibility. For this purpose, an aliquot of the pool was incubated with 100 mmol/l glucose during 1–7 days. Another aliquot was incubated at 37°C with glucose concentrations ranging from 13 to 100 mmol/l during 4 days, a period roughly equal to LDL mean life in plasma. Fructosamine was determined in LDL fractions using Nitro Blue Tetrazolium (NBT) reagent and serum con* Corresponding author. Fax: +54-1-823-7351; e-mail: ssanguinetti@dbc.ffyb.uba.ar.

Influence of the medium on the assessment of LDL resistance to oxidation: lag time in phosphate buffered saline is longer than in sodium chloride solution

It is well known that oxidative modification of human low density lipoprotein (LDL) plays an important role in the atherogenic process [1,2]. Previously published data indicate that there is a relationship between LDL in vitro susceptibility to oxidation and atherosclerotic risk in humans [3]. LDL in vitro oxidation can be mediated by cells or mimicked in a cell free system with transition metal ions like iron or copper, as prooxidants. One of the procedures to determine the susceptibility of LDL to in vitro oxidation consists in continuously monitoring the increase in absorbance at 234 nm due to the formation of conjugated fatty acid hydroperoxides that result from lipid peroxidation. The kinetics of copper-induced LDL oxidation shows a lag phase which ends when LDL is depleted of its antioxidants and the rate of lipid peroxidation is rapidly accelerated. The lag time is related not only to antioxidants content but also to LDL lipid components such as polyunsaturated fatty acids and triglycerides [4]. Moreover, the lag time also depends on the conditions selected to promote copper oxidation [5,6]. The most important factors are: LDL and Cu concentration, Cu/LDL ratio, temperature and medium. Usually, oxidation is assessed in phosphate buffered saline (PBS): 0.01 M phosphate, 0.16 M NaCl, pH 7.4. Although it is known that PBS affects iron redox potential through iron chelation [7], the effect of PBS on copper is not clearly understood. We have compared copper-induced LDL peroxidation in PBS and in 0.16 M NaCl, pH 7.4 saline solution (SS). We chose a group of subjects with a wide range of lag time. Twenty subjects, 11 males and 9 females, (aged 25–70) were studied. LDL was isolated from fasting plasma supplemented with EDTA (1 g/l), by sequential flotation ultracentrifugation within d= 1.019–1.063 g/ml. EDTA was present throughout all the steps of the isolation procedure. LDL was extensively dialyzed in the dark at 4°C against SS. In order to carry out oxidation experiments, an aliquot of EDTA-free LDL was adjusted to 0.1 mg LDL protein/ ml in PBS, and the other fraction of LDL was diluted to the same concentration using SS. Oxidation was initiated by addition of freshly prepared CuCl2 ·2H20 solution (final concentration 10 mM). The kinetics of LDL oxidation was determined by monitoring the change in absorbance at 234 nm using a Hitachi U1100 spectrophotometer (Tokyo, Japan), at 37°C. Absorbance was registered every 5 min for at least 4 h. The pH was controlled along peroxidation and as with PBS, it also remained stable in the SS preparation. The initial absorbance at 234 nm was subtracted from each * Corresponding author. Fax: +54 1 9618533.