Regina L. W Wikinski

Ischemic postconditioning reduces infarct size by activation of A1 receptors and K+ATP channels in both normal and hypercholesterolemic rabbits

The effect of ischemic postconditioning (Postcon) in hypercholesterolemic animals is unknown. The objectives were to determine if ischemic preconditioning (IPC) and Postcon reduce infarct size in hypercholesterolemic animals and to assess if A1 receptors and K+ATP channels are involved in Postcon mechanisms. Isolated rabbit hearts were perfused according to the Langendorff technique and subjected to 30 minutes of ischemia and 30 minutes of reperfusion (G1). In Group 2, IPC was performed (1 cycle of 5 minutes ischemia/reperfusion) before 30 minutes of ischemia. In Group 3 (G3), Postcon was performed (2 cycles of 30-second reperfusion/ischemia) after 30 minutes of ischemia. The G3 protocol was repeated in G4 and G5, but during Postcon, an A1 receptor blocker (DPCPX, 200 nM) and glybenclamide (K+ATP, blocker, 0.3 μM) were administered, respectively. The G1 to G5 protocols were repeated in animals fed with an enriched cholesterol diet (1%) for 4 weeks (G6 to G10). The infarct size was measured by triphenyltetrazolium. The infarct size was 16.6 ± 4.6% in G1 and 25.8 ± 7.3% in G6, and IPC and Postcon reduced the infarct area in both normal and hypercholesterolemic animals (G2: 5.1 ± 1.7% [P < 0.05] and G3: 5.4 ± 0.9% [P < 0.05] in normal animals; G7: 4.1 ± 1.6% [P < 0.05] and G8 4.8 ± 0.9% [P < 0.05], in hypercholesterolemic animals). Both DPCPX and glybenclamide abolished the effect reached by Postcon. Thus, Postcon reduces infarct size in normal and hypercholesterolemic animals through the activation of A1 and K+ATP channels.

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.

LIPOPROTEIN ALTERATIONS, ABDOMINAL FAT DISTRIBUTION AND BREAST CANCER

Plasma lipid profile and abdominal obesity have been associated with breast cancer risk, however published results have been inconsistent. To clarify these associations we studied lipid and lipoprotein alterations, obesity degree and body fat distribution, in 30 newly diagnosed breast cancer patients without treatment and 30 controls matched by age and menopausal status. Both pre and postmenopausal breast cancer patients presented higher body mass index, waist/hip ratio and insulin levels than their matched controls. An increase in triglycerides and a decrease in HDL‐cholesterol, especially in the HDL2 subfraction, were observed in patients with breast cancer. Besides, HDL particle from these patients showed increased apo A1/HDL‐cholesterol ratio. These alterations were correlated with waist/hip ratio.

Abnormal capacity to induce cholesterol efflux and a new LpA-I pre-β particle in type 2 diabetic patients

In this study, we first characterized the lipoprotein components of serum samples obtained from a group of well-controlled diabetic patients and from healthy subjects in fasting and postprandial states. We then explored some aspects of reverse cholesterol transport in the same population. Patients showed high levels of fasting triglycerides, postprandial triglyceride responses and LpC-III levels (3.18+/-0.86 vs 2.17+/-0.54 mg/dl, P < 0.001). There were also positive correlations between LpC-III and fasting triglycerides (r = 0.82, P < 0.001), total triglyceride area (r = 0.75, P < 0.001) and incremental triglyceride area (r = 0.54, P < 0.001). HDL-C and apo A-I were significantly decreased in diabetic patients due to a selective reduction in LpA-I subfraction, whose antiatherogenic role is generally accepted (37.4+/-8.0 vs 49.2+/-12.5 mg/dl, P < 0.001). In addition, HDL from patients proved to be triglyceride enriched and cholesteryl ester depleted, alterations which were further amplified in the postprandial state. The molar ratio HDL-C/apo A-I + apo A-II, already defined as a predictor of apo A-I fractional catabolic rate, was significantly diminished in the patient group (15.1+/-2.2 vs 20.8+/-3.3, P < 0.001), thus suggesting an accelerated catabolism of apo A-I. For the first time, we describe here the presence of a small apo A-I-containing particle, isolated by two-dimensional electrophoresis and characterized by immunoblotting, only in samples from diabetic patients. This particle that we named pre-beta0, has an apparent molecular weight of 40 kDa. As regards the capacity of serum samples to promote cholesterol efflux from [3H]cholesterol-labeled Fu5AH rat hepatoma cells, patient samples were found to induce significantly lower cholesterol efflux than controls only in the postprandial state (21.2+/-3.3 vs 23.8+/-1.8%, P = 0.012). The presence of pre-beta0 in samples from diabetic patients might therefore be associated to an altered capacity of these serum samples to promote cellular cholesterol efflux. Overall, these abnormalities may contribute to a delay in the reverse cholesterol transport pathway in type 2 diabetic patients.

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.

New mutations in the lipoprotein lipase gene in a young boy with chylomicronaemia syndrome and in his family.

The case is reported of a 4‐y‐old boy with chylomicronaemia syndrome, under treatment with a low‐fat diet and medium‐chain triglycerides. The clinical and biochemical characteristics of the patient and 11 members of his family were studied. Lipoprotein profile, lipoprotein lipase (LPL) mass and activity were evaluated. Nucleotide substitutions in LPL promoter and exons were screened. The proband presented with severe hypertriglyceridaemia (triglycerides = 13.25 mmol l−1) and non‐detectable LPL activity and mass. The boy was a compound heterozygote for four molecular defects in the LPL gene, two of which have not been reported before (C±T764± C±T/Arg170± Leu; GG±1482± GG±Gly409± Gly). Among the family members, the proband was the only one who carried two genetic variants that modify LPL amino acid composition.

HDL metabolic activities in a boy with lipoprotein lipase deficiency and his family

Background  Lipoprotein lipase (LPL) deficiency is a rare autosomal recessively inherited disease characterized by elevated triglyceride, low total cholesterol and quantitative and qualitative alterations of high‐density lipoprotein (HDL). The aim of the present study was to explore HDL metabolic activities in a patient with LPL deficiency and in his family (n = 11).