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.

Abnormal capacity to induce cholesterol efflux and a new LpA-I pre-beta 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.

Evaluation of the blood components considered as risk predictors in coronary heart disease.

The study included 249 patients two days before cardiovascular surgery and 73,915 control subjects. Results obtained were analyzed by grouping the individuals according to sex and age. In coronary heart disease (CHD) in males, total cholesterol was found higher than in controls (mean +/- D.S.: 241.9 +/- 44.7 vs 223.6 +/- 43.0 mg/dl, p < 0.01) between 25 and 49 years of age, this significance being lost with age. Triglycerides were also higher (197 +/- 107.3 vs 161.6 +/- 97.7 mg/dl, p < 0.01) in the CHD male population between ages 25 and 69. In CHD females, triglycerides were higher (116.9 +/- 56.2 vs 91.5 +/- 43.3 mg/dl, p < 0.05) between ages 25 and 49; cholesterol showed no difference at any of the ages studied. HDL-C was much lower in both sexes of CHD patients at all ages studied (p < 0.001). Uric acid was higher in CHD males between ages 25 and 49 (p < 0.05), this significance being lost in the older age CHD group. Other components such as glycated hemoglobin, glucose and ionized calcium, were not different from those of the control group.

[Partial lecithin-cholesterol acyltransferase (LCAT) deficiency syndrome]. / Síndrome de deficiencia parcial de lecitina-colesterol aciltransferasa (LCAT).

This syndrome is a pathological entity of low incidence which mainly affects high density lipoprotein (HDL) metabolism. We here show the first case reported in our country, observed in a 63-year-old woman who showed bilateral corneal opacity and eruptive xanthomas in both arms. The lipoprotein profile disclosed severe hypertriglyceridemia and normocholesterolemia, although the percentage of cholesteryl esters was low. Plasma levels of HDL-cholesterol and HDL major apolipoproteins, A-I and A-II, were markedly decreased. The patient also showed glucose intolerance and hematological alterations related to abnormal lipid composition of erythrocyte membranes. As evaluated by the exogen substrate method, LCAT activity proved to be 82% lower in the patient than in a control subject. It is noteworthy that the patient had experienced cardiac events and presented hypertension, neither of which has been commonly documented in partial LCAT deficiency syndromes.

[Low density lipoprotein rich in triglycerides and hepatic lipase activity in insulin-dependent diabetic patients]. / Lipoproteína de baja densidad rica en triglicéridos y actividad de lipasa hepática en pacientes diabéticos insulino-dependientes.

Genetic hepatic lipase (HL) deficiency is associated with low density lipoprotein (LDL) rich in triglycerides (TG), whose affinity for B:E receptors is decreased. In rats, experimental hypoinsulinemia produces HL deficiency. However, the relation between human insulin-dependent Diabetes Mellitus (IDDM), HL activity and the characteristics of LDL have not been studied. The objective of our study is to evaluate the relation between HL activity and the chemical composition of LDL in treated IDDM patients. Subjects were 15 IDDM patients and 15 controls (C), matched for sex and body mass index (BMI). The IDDM patients were classified by the WHO criteria, were free of nephropathy and hypothyroidism, and received no medication except insulin. Controls were clinically healthy and normolipidemic with no family history of diabetes. The IDDM group was divided into two subgroups: subgroup IDDM-A (n = 9) with HL values > or = 4.3 and IDDM-B (n = 6) with HL < or = than 4.2 mumoles glycerol/ml h. the HL in IDDM was lower than in C (p < 0.001). Table 1 shows clinical data. Blood samples were drawn after 12 h fasting. Percentage of HbA1c and plasma concentrations of glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol and TG were assayed. LDL was separated by sequential ultracentrifugation at densities of 1.019-1.063 g/ml and its chemical composition was analyzed. The most relevant results were: plasma TG concentration was higher in IDDM than in C (p < 0.05) (Table 2), although average values DMID not exceed the reference values of 200 mg/dl. The TG-LDL were higher in IDDM than in C: 24.8 +/- 2.7 vs 17.5 +/- 1.1 mg/dl plasma, media +/- SE, (p < 0.02). This difference reflected the values of IDDM-B, whose plasma concentrations of TG-LDL were higher than in C: 32.3 +/- 3.6 vs 17.5 +/- 1.1 mg/dl (p < 0.001), and also higher than in IDDM-A (p < 0.02). (Table 3). The chemical composition of LDL in IDDM-B contained a higher percentage of TG than C: 8.5 +/- 0.7 vs 6.8 +/- 0.3% (p < 0.05), a lower percentage of cholesterol than IDDM-A: 39.0 +/- 1.7 vs 45.2 +/- 2.2% (p < 0.05) and also a larger percentage of proteins than IDDM-A: 28.9 +/- 1.9 vs 20.8 +/- 1.0% (p < 0.01). The correlations between TG/cholesterol and HL activity in IDDM were r = -0.53 (p < 0.05) and in IDDM-B, r = -0.81 (p = 0.05). The noteworthy result of this study is the modification of the LDL particle in IDDM, rich in TG in patients with low HL activity. Anomalies in the chemical composition of LDL like those described decrease the uptake of this particle by its physiological B:E receptors. It has recently been demonstrated that LDL is an indisoluble association of lipids and apoproteins, and that both act simultaneously to hold the apoB in a spatial position that expresses normal epitopes. It has been described that particles of LDL rich in TG and poor in cholesterol, shows low affinity for LDL receptors in human fibroblasts. Also in IDDM the interaction of LDL rich in TG with B:E receptors is decreased. This might be one more mechanism contributing to the accelerated atherosclerosis of these patients. Our results suggest that there may be a threshold of HL activity for the complete hydrolysis of the TG of LDL, for the normalization of the TG/cholesterol relation and for the conformation of typical LDL particles.

[Acquired partial lipodystrophy. Insulin resistance, hepatic lipase activity and small and dense LDL particles]. / Lipodistrofia parcial adquirida. Resistencia insulínica, actividad lipasa hepática y partículas LDL pequeñas y densas.

Partial lipodystrophy (PLD) is an infrequent condition characterized by symmetric loss of subcutaneous adipose tissue in the upper or lower part of the body, although occasionally it affects only the extremities. In all cases it appears along with acantosis nigricans (AN), insulin resistance and impairment in the metabolism of lipids and carbohydrates. The case depicted pertains to a 49 year old female with no family history involving loss of adipose tissue in face and upper body. No fat in lower part of body was observed. The patient showed facial thinning at age 8, AN at 11 and gestational diabetes during her fourth pregnancy at 33. At present, the patient presents severe hyperglycemia and hyperinsulinemia with a marked insulin resistance. Type IV hyperlipoproteinemia (OMS), declined C-HDL and Apo A1 and low C-LDL but with a high proportion of small and dense LDL particles were present. Non esterified fatty acids were high. Lipoprotein lipase and hepatic lipase activities are in the lower limit and increased respectively. Fraction C3 of the complement was diminished. No mutations were observed either in codons 170, 809 and 972 of the IRS-1 receptor or in codon 276 of the adrenergic beta 2 gene.

New method for isolating and quantifying intermediate and beta-very-low-density lipoprotein cholesterol.

We describe a new method, useful to clinical laboratories, for assessing intermediate density (IDL) or beta-very-low-density (beta-VLDL) lipoprotein cholesterol. The technique involves selective precipitation properties of the qualitative Wieland and Seidel post-electrophoretic method that immobilizes IDL and beta-VLDL in the beta-zone of an agarose slide (Clin Chem 1973;19:1139-41). In our method, we separate low-density lipoprotein (LDL) in a second electrophoretic step, in which LDL moves toward the anode, and then quantify the cholesterol of the above lipoproteins remaining in the precipitate band at the beta-zone. Replicate within-run precision (CV) of 15 aliquots of a sera pool was 10.1%. The correlation with sequential ultracentrifugation of 30 samples was r = 0.96 (P less than 0.001). Serum reference values for 30 normal individuals are 57 +/- 7.0 mg/L. Seven phenotype III hyperlipoproteinemic patients had the highest concentrations of IDL or beta-VLDL cholesterol in serum, 1620 +/- 346 mg/L.

Alterations in dense LDL subfractions in normolipidemic IDDM patients.

Low density lipoproteins (LDL) of human plasma, consist of a continuum of particles subclasses with distinct physicochemical, immunological and hydrodynamic characteristics. Such structural differences are intimately linked to atherogenesis. The current study was designated to investigated the LDL subclasses profile in 12 normolipidemic IDDM patients, and compare it with 11 healthy controls. Four plasma LDL subfractions were isolated by sequential ultracentrifugation in the density range (1.025-1.063 g/ml) and were characterized by their content of free and esterified cholesterol, triglycerides, phospholipids, and proteins. The net electrical charge was evaluated. Plasma concentration of the two denser LDL subfractions were higher in IDDM patients vs control subjects, due to an increase in cholesterol (free and esterified) and phospholipids, while more buoyant subfractions in the two groups were not different. In IDDM patients the LDL profile was skewed towards the dense subclasses LDL-III and LDL-IV, being significant this increase for LDL-IV: 22.3 +/- 5.2 vs 18.3 +/- 4.0%, p < 0.05, X +/- DS. In the healthy controls the LDL profile was skewed toward the lighter subclasses (LDL-I and LDL-II), being significant for LDL-II: 30.0 +/- 4.3 vs 23.3 +/- 4.2%, p < 0.005. Diabetic patients, even those who are normolipidemic, present increased risk of premature atherosclerosis. This suggest that normal values in lipid and lipoprotein profile can mask deeper alterations, such as changes in the composition and distribution of the denser subclasses, whose characteristics make them potentially more atherogenic. Despite the apparently normolipidemic status, dense LDL particles considered to be atherogenic, are increased in IDDM patients.

Glucose and orthophosphate incorporation and lactate release in the perfused hind limb of the rat during lactic acidemia.

A perfused preparation of the hind limb of normal and diabetic rats was used to study the effects of lactic acidosis, alone or associated with hypoinsulinemic diabetes, on the incorporation of glucose and inorganic orthophosphate (Pi) into the skeletal muscle. A well oxygenated perfusate was recirculated for ninety minutes during which the lactic acid accumulated into the medium with the ensuing pH drop. The perfusions were practiced in the hind limb of alloxanized diabetic rats, in the hind limb of diabetic rats with perfusate containing 200 microU of insulin/ml, in the hind limb of 24 hour fasted rats, and on the hind limb of fed rats, and they were compared to similar groups with normalized pH perfusate with a sodium bicarbonate infusion. In the diabetic perfusions with lactic acidemia, it was observed that the addition of insulin increased the uptake of Pi and of glucose, and reduced the release of Pi by the muscular tissues. A smaller release of Pi by the preparations obtained from fed rats was observed when compared to the hind limb preparations of fasted rats. The diabetic preparations showed an increased glucose uptake when the pH was normalized, and a decrease of Pi released by the muscles, even in the absence of insulin, and at the same time, the administration of insulin associated with the normalization of pH increased the uptake of Pi and of glucose, and decreased the Pi released by the muscles. In all the groups, the administration of sodium bicarbonate significantly increased the lactate release into the medium. It was also found that the lactic acidosis reduced the uptake of Pi by the preparations inducing hyperphosphatemia. According to these results, muscular tissue plays a role in the hypophosphatemia that has been reported in the insulin treated diabetic ketoacidosis by increasing the incorporation of Pi and reducing its release by the same tissue.

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. The association between lipoprotein alterations and abdominal obesity independent of menopausal status, in untreated newly diagnosed breast cancer patients is reported for the first time in this study.

HDL oxidability and its protective effect against LDL oxidation in Type 2 diabetic patients.

Low density lipoprotein (LDL) oxidation is a crucial step in the atherosclerotic process. High density lipoprotein (HDL)-associated enzymes such as paraoxonase could exert a protective effect on LDL oxidation in the arterial wall, an effect which could be impaired in Type 2 diabetes mellitus (T2DM). We studied copper-induced oxidation in LDL and HDL isolated from 17 T2DM patients with fair glycaemic control and HDL-cholesterol within normal range and 17 healthy normolipidaemic control subjects. To evaluate the effect of HDL on LDL oxidation in diabetic and control subjects, we assessed copper-induced oxidation in HDL/LDL mixtures, with each lipoprotein isolated from the same subject. Relationships with HDL chemical composition, alpha-tocopherol content and serum paraoxonase activity were investigated. Oxidation was promoted by lipoprotein incubation with copper and then thiobarbituric acid reactive substances (TBARS), conjugated diene production and electrophoretic mobility in agarose gel were measured. In T2DM subjects HDL oxidation was higher than in controls. However, HDL from diabetics was as effective as control HDL to inhibit LDL oxidation. Neither HDL chemical composition nor serum paraoxonase activity showed any difference as compared to control subjects. In contrast, HDL from T2DM subjects showed a higher alpha-tocopherol content which positively correlated with HDL oxidability. Paraoxonase activity positively and strongly correlated with HDL inhibitory effect on LDL oxidation in patients and controls belonging to the heterozygous activity phenotype. Besides, LDL oxidability showed no differences between patients and controls. These results suggest that fairly-controlled T2DM patients with HDL-cholesterol levels within normal range show: 1) normal HDL ability to inhibit LDL oxidation related to normal paraoxonase activity; 2) higher HDL oxidability in spite of its high alpha-tocopherol content, which could favour tocopherol-mediated peroxidation and 3) normal LDL oxidability possibly due to the lack of significant lipoprotein structural alterations.

Alterations in dense LDL subfractions in normolipidemic IDDM patients

Low density lipoproteins (LDL) of human plasma, consist of a continuum of particles subclasses with distinct physicochemical, immunological and hydrodynamic characteristics. Such structural differences are intimately linked to atherogenesis. The current sludy was designated to investigate the LDL subclasses profile in 12 normolipidemic IDDM patients, and compare it with 11 healthy controls. Four plasma LDL subfractions were isolated by sequential ultracentrifugation in the density range (1.025- 1.063 g/ml) and were characterized by their content of free and esterified cholesterol, triglycerides, phospholipids, and proteins. The net electrical charge was evaluated. Plasma concentration of the two denser LDL subfractions were higher in IDDM patients vs control subjects, due to an increase in cholesterol (free and esterified and phospholipids, while more buoyant subfractions in the two groups were not diferent. In IDDM patients the LDL profile was skewed towards the dense subclasses LDL-III and LDL-IV, being significant this increase for LDL-IV: 22.3 ñ 5.2 vs 18.3 ñ 4.0 per cent, p<0.05, XñDS. In the healthy controls the LDL profile was skewed toward the lighter subclasses (LDL-I and LDL-II), being significant for LDL-II: 30.0 ñ 4.3 vs 23.3 ñ 4.2 per cent, p<0.005. Diabetic patients, even those who are normolipidemic, present increased risk of premature atherosclerosis. This suggest that normal values in lipid and lipoprotein profile can mask deeper alterations, such as changes in the composition and distribution of the denser subclasses, whose characteristics make them potentially more atherogenic. Despite the apparently normolipidemic status, dense LDL particles considered to be atherogenic, are increased in IDDM patients.