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.

AIMS/HYPOTHESIS:Chemical and biological characteristics of LDL(-) from type 1 diabetic subjects were analysed. The diabetic patients were studied during poor and optimised glycaemic control.MATERIALS AND METHODS:Total LDL was subfractionated into electropositive LDL(+) and electronegative LDL(-) by anion exchange chromatography and the lipid and protein composition of the two determined.RESULTS:LDL(-) differed from LDL(+) in that it had higher triglyceride, non-esterified fatty acids, apoE, apoC-III and platelet-activating factor acetylhydrolase (PAF-AH), as well as lower apoB relative content. No evidence of increased oxidation was observed in LDL(-). LDL(-) increased two-fold the release of interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in endothelial cells, suggesting an inflammatory role. Optimisation of glycaemic control after insulin therapy decreased the proportion of LDL(-), but did not modify the composition of LDL subfractions, except for a decrease in PAF-AH activity in LDL(-). The possibility that LDL(-) could be generated by non-enzymatic glycosylation was studied. Fructosamine and glycated LDL content in LDL subfractions from type 1 diabetic patients was greater than in LDL subfractions isolated from normoglycaemic subjects, and decreased after glycaemic optimisation in both subfractions. However, no difference was observed between LDL(+) and LDL(-) before and after insulin therapy.CONCLUSIONS/INTERPRETATION:These results provide evidence that LDL(-) is not produced by glycosylation. Nevertheless, LDL(-) from diabetic patients displays inflammatory potential reflected by the induction of chemokine release in endothelial cells. This proatherogenic effect could be related to the high PAF-AH activity in LDL(-).

Electronegative low-density lipoprotein (LDL(−)) is a plasma-circulating LDL subfraction with proinflammatory properties that induces the production of chemokines in cultured endothelial cells. However, the specific mechanism of LDL(−)-mediated chemokine release is presently unknown. A characteristic feature of LDL(−) is an increased content of lysophosphatidylcholine (LPC) and non-esterified fatty acids (NEFA). The effect of increasing amounts of LPC and NEFA associated with LDL on the release of chemokines by endothelial cells was studied. Total LDL was subfractionated by anion-exchange chromatography in electropositive (LDL(+)) and LDL(−). LDL(−) contained two-fold more LPC and NEFA than LDL(+) and induced two- to four-fold more (p < 0.05) interleukin-8 (IL-8, 11.5 ± 8.2ng/105 cells) and monocyte chemotactic protein-1 (MCP-1, 10.8 ± 3.8ng/105 cells) release by human umbilical vein endothelial cells (HUVEC) than LDL(+) (IL-8: 3.4 ± 1.5ng/105 cells, MCP-1: 5.8 ± 2.9ng/105 cells). The content of LPC and NEFA in LDL(+) was increased by enzymatic treatment with secretory phospholipase A2 (sPLA2) at 5ng/mL or 20ng/mL or by incubation with NEFA at 2mmol/L. Modification of LDL(+) by both methods did not result in oxidative modification as demonstrated by the lack of change in antioxidants, conjugated dienes and malondialdehyde content. sPLA2 treatment resulted in an increase in LPC and NEFA in LDL(+) which enhanced its ability to release IL-8 and MCP-1 by HUVEC in a concentration-dependent manner (sPLA2(5)-LDL; IL-8: 7.1 ± 3.8ng/105 cells, MCP-1: 8.0 ± 5.1ng/105 cells; sPLA2(20)-LDL; IL-8: 20.8 ± 11.2ng/105 cells, MCP-1: 15.0 ± 7.5ng/105 cells). NEFA loading of LDL(+) also favored the release of IL-8 and MCP-1 (IL-8: 7.8 ± 6.1ng/105 cells, MCP-1: 8.4 ± 2.7ng/105 cells, p < 0.05 versus LDL(+)). These effects were observed when modified LDL(+) reached a content of LPC and/or NEFA similar that of LDL(−). These data indicate that non-oxidized polar lipids associated with LDL promote an inflammatory response in endothelial cells and suggest that increased NEFA and LPC could be involved in the inflammatory activity of LDL(−).

Subclinical hypothyroidism (SH) is a frequent condition that may be associated with increased cardiovascular risk. There is current interest in determining the effect, if any, of substitutive therapy with l-thyroxine (L-T4) on cardiovascular risk factors in SH and, particularly, on those associated with emerging cardiovacular risk, such as apolipoprotein (apo) B, lipoprotein (Lp) (a), total homocysteine (t-Hcy), and C-reactive protein (CRP). Thus, the aim of this study was to assess the impact of euthyroidism restoration on these emerging risk factors in SH. Forty-two patients diagnosed with SH were consecutively recruited before treatment. These patients were treated with L-T4 for 3 to 6 months with the dose necessary to restore euthyroidism. Lp(a), fasting and postmethionine (n = 28) t-Hcy, and CRP did not change with substitutive therapy, regardless of the respective baseline values, and the decrease in apo B paralleled that of low-density lipoprotein (LDL) cholesterol. Similarly, no treatment effect was observed on homocysteine or CRP in patients with thyrotropin-stimulating hormone (TSH) >10 mIU/L. Monitoring of emerging risk factors did not offer additional arguments for treating patients with SH and, thus, is not justified in their clinical management.

La LDL electronegativa (LDL[–]) es una fracción minoritaria de LDL plasmática con características aterogénicas, como menor afinidad por el receptor de LDL e inducción de liberación de quimiocinas en células endoteliales. Por otra parte, la LDL(–) presenta un aumento en el contenido en ácidos grasos no esterificados (NEFA) y lisofosfatidilcolina (LPC), que son productos de la acción de la fosfolipasa A2 (PLA2) sobre la LDL. Por ello, el objetivo de este trabajo fue estudiar la implicación de la PLA2 en la generación de LDL(–). Se modificó in vitro LDL con PLA2 (PLA2-LDL) a diferentes concentraciones (0, 0,5, 1, 5, 10 y 20 μg/l) durante 2 h a 37 °C en presencia de albúmina. Se encontraron similitudes entre LDL(–) y PLA2-LDL en sus propiedades. El tratamiento de la LDL con PLA2 indujo un incremento progresivo en la carga eléctrica negativa, un menor tamaño, una menor susceptibilidad a la oxidación y mayor a la agregación, y un contenido aumentado en NEFA y LPC. En su interacción con el receptor de LDL en fibroblastos, la PLA2-LDL presentó una afinidad disminuida. Por otra parte, también indujo la liberación de MCP-1 e IL-8 en células endoteliales. Todos los efectos de la PLA2-LDL fueron dependientes de la dosis y similares a los de la LDL(–) cuando la PLA2-LDL presentó un contenido en NEFA y LPC semejante al de la LDL(–) (la LDL tratada con 1-5 μg/l de PLA2). La modificación de la LDL por acción de la PLA2 podría implicar la generación de LDL(–), ya que existen coincidencias tanto fisicoquímicas como biológicas entre la LDL(–) y la PLA2-LDL. Electronegative LDL (LDL[–]) is a minor plasma LDL fraction with atherogenic characteristics such as lower LDL receptor affinity and induction of chemokine release in endothelial cells. On the other hand, LDL(–) shows an increase in the content of non-esterified fatty acids (NEFA) and lysophosphatidylcholine (LPC), products of the action of phospholipase A2 (PLA2) on LDL. For this reason, the aim of this work was to study the relationship of PLA2 in LDL(–) generation. LDL was modified in vitro with PLA2 (PLA2-LDL) at different concentrations (0, 0.5, 1, 5, 10 y 20 μg/l) for 2 h at 37 °C in the presence of albumin. Similarities were found between LDL(–) and PLA2-LDL properties. Treatment with PLA2 induced a progressive increase in the negative charge of LDL, decreased size, diminished susceptibility to oxidation and greater to aggregation, and higher content in NEFA and LPC. PLA2-LDL presented decreased affinity in its interaction with the LDL receptor in fibroblasts. Furthermore, it also induced MCP-1 and IL-8 release by endothelial cells. All the PLA2-LDL effects were dose-dependent and similar to those of LDL(–) when PLA2-LDL (LDL treated with 1-5 μg/l of PLA2) presented a NEFA and LPC content similar to that of LDL(–). LDL modification with PLA2 could be responsible for LDL(–) generation, since physico-chemical and biological coincidences exist between LDL(–) and PLA2-LDL.

El posible papel de los folatos en la prevención de enfermedades cardiovasculares o neurodegenerativas es un tema de actualidad. Diversos estudios epidemiológicos han demostrado una asociación inversa entre los valores de esta vitamina en sangre y la morbimortalidad cardiovascular o la enfermedad de Alzheimer. Múltiples estudios han constatado los efectos hipohomocisteinemiantes del folato. Recientemente se han descrito efectos beneficiosos del folato en el endotelio que son homocisteínaindependientes. Para evaluar su potencial relevancia fisiopatológica se requiere que haya modelos en que los que se pueda disociar hiperhomocisteinemia y deficiencia de folato. Estudio histológico y morfométrico de las lesiones arterioscleróticas en ratones C57BL/6 alimentados con dieta Western (una dieta con un alto contenido en grasas saturadas) deficiente o no en folatos. Los animales con déficit vitamínico de folato no presentaron hiperhomocisteinemia. Tampoco presentaron diferencias en la glucosa ni en los parámetros lipídicos, hematológicos o la función renal. La deficiencia en folatos no aumentó el área (5.310 ± 1.346 frente a 4522 ± 1276 mm2 en el grupo control) ni el número de lesiones arterioscleróticas (1,4 ± 0,3 frente a 2,0 ± 0,5 en el grupo control). El ratón es un buen modelo para diferenciar los efectos debidos a déficit de folato de los ocasionados por hiperhomocisteinemia, un binomio que suele ser inseparable en humanos. La deficiencia de folato, en ausencia de hiperhomocisteinemia, no incrementa la magnitud ni el número de lesiones arterioscleróticas en el ratón. Introduction and objectives. The potential role of folates in the prevention of cardiovascular and neurodegenerative diseases is a topic of current interest. Several studies have showed an association between these vitamin levels and cardiovascular and Alzheimer morbidity and/or mortality. Many studies have demonstrated the homocysteinelowering effects of folates. It has recently been demonstrated that folates have homocysteineindependent protective actions at endothelial level. To evaluate their pathophysiological significance, models are required in which folate deficiency and hyperhomocysteinemia can be dissociated.The potential role of folates in the prevention of cardiovascular and neurodegenerative diseases is a topic of current interest. Several studies have showed an association between these vitamin levels and cardiovascular and Alzheimer morbidity and/or mortality. Many studies have demonstrated the homocysteinelowering effects of folates. It has recently been demonstrated that folates have homocysteineindependent protective actions at endothelial level. To evaluate their pathophysiological significance, models are required in which folate deficiency and hyperhomocysteinemia can be dissociated. Histologic and morphometric study of aortic atherosclerosis was conducted in C57BL/6 mice fed a Western type diet (rich in saturated fat) containing or not folates. Animals with vitamin deficiency did not present hyperhomocysteinemia. No changes were observed in lipid profile, glucose or hematological and renal function parameters. Folate deficiency did not increase either the size (5310 ± 1346 mm2 versus 4522 ± 1276 mm2 in the control group) or number of atherosclerotic lesions (1.4 ± 0.3 versus 2.0 ± 0.5 in the control group). The mouse is a good model for separating the differential effects of folate deficiency from those of hyperhomocysteinemia. Folate deficiency, in absence of hyperhomocysteinemia, does not increase either the size or number of atherosclerotic lesions.

To assess postprandial lipidemia in normotriglyceridaemic type 2 diabetic patients treated with diet only, 12 non-obese patients (8 males, hemoglobin A1c [HbA1c] 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.

The immediate effects of intense aerobic exercise on the composition and oxidizability of low- (LDL) and high-density lipoproteins (HDL) were studied in 11 male athletes. Plasma parameters known to affect lipoprotein oxidizability were also evaluated. Lipophilic antioxidants, including α-tocopherol and carotenoids, paraoxonase and malondialdehyde (MDA) in plasma remained unchanged after exercise. Increases in the concentration of uric acid, bilirubin and ascorbic acid after the race resulted in a significant increase in total antioxidant serum capacity. LDL, but not HDL, increased its 'in vitro'-induced susceptibility to oxidation and the proportion of electronegative LDL (LDL(−)). The ability of HDL to inhibit the oxidation of LDL remained unchanged after exercise. The enhanced oxidizability of LDL was not explained by increments in its aldehyde content or by decrements in antioxidants. The major compositional change in LDL was an increase in non-esterified fatty acid (NEFA) content (from 4.00±1.24 to 19.00±14.18 mol NEFA/mol apoB). NEFA also increased in plasma and HDL. 'In vitro' experiments showed that incubation of LDL with increasing amounts of NEFA induced a concentration-dependent increase in the proportion of LDL(−). Moreover, a slightly increased NEFA content in LDL (15–50 mol NEFA/mol apoB) induced higher susceptibility to oxidation. These 'in vitro' results concur with those observed in LDL obtained from athletes after exercise, i.e. a concentration of approximately 20 mol NEFA/mol apoB increased LDL oxidizability and LDL(−) proportion. We conclude that changes in the qualitative characteristics of LDL after exercise were unrelated to oxidative stress, but were related to the increase in LDL-associated NEFA content.

Abstract The determination of the total concentration of plasma homocysteine is of interest in a variety of clinical circumstances, especially, in the evaluation of the risk of cardiovascular disease. However, most of the methods available to date, many of them chromatographic, are not well suited for the majority of clinical laboratories. Several automated methods are now or will be, shortly, commercially available. We have compared one of them, the fluorescence polarization immunoassay (FPIA) adapted to the IMx® analyzer (Abbott Laboratories), with the high-performance liquid chromatography (HPLC) method with fluorescent detection currently used in our laboratory. The results show that the FPIA-IMx® method is less imprecise and slightly more sensitive than the HPLC. The comparison of 67 clinical plasma specimens indicated that there is a proportional error disagreement between FPIA-IMx® and HPLC (FPIA=1.19 HPLC + 0.92; confidence region for slope and y-intercept were, respectively, from 1.06 to 1.31 and from−0.06 to 2.32). The nature of this error is not explained by the experiments performed to study the inaccuracy of both methods, which included the investigation of dilution parallelism, analytical recovery and cross-reactivity. The different results of homocysteine concentration obtained with FPIA-IMx® and HPLC must be taken into account when a change of methodology is under consideration.

MTHFR C677T and A1298C polymorphisms have been reported to be associated with the risk of myocardial infarction (MI), although the results of previous studies have been inconsistent. The aim of this study was to explore whether these polymorphisms play a role in the genetic susceptibility to MI. A comprehensive search of MEDLINE and EMBASE databases was conducted for studies evaluating the association between the C667T and A1298C polymorphisms and MI risk. Odds ratios (OR) with 95% confidence intervals (CIs) were calculated to assess the strength of association in the dominant model, recessive model, allelic model, and genotypes contrast. A total of 47 studies were finally included in this meta-analysis. Overall, the results showed no statistically significant association between C667T and A1298C polymorphisms and MI risk. However, in subgroup analysis by ethnicity, the T allele of C677T polymorphism was associated with a 63% increased risk of MI compared with the C allele (T vs. C, OR = 1. 63, 95%CI = 1.15–2.10, fixed effects) in African populations, while compared to wild homozygote genotype, CT genotype was associated with a decreased risk of MI in North American populations (CT vs. CC, OR = 0.81, 95%CI = 0.64–0.98, fixed effects). Moreover, C677T polymorphism had a protective effect against MI risk under the dominant model (OR = 0.93, 945%CI = 0.87–0.99, fixed effects) in elderly (≥ 50) population. The A1298C polymorphism was not significantly associated with MI risk. Unlike A1298C polymorphism, C677T polymorphism was associated with risk of MI in African, North American, and elderly populations.

Hyperhomocyst(e)inemia is an independent risk factor for atherothrombosis in several clinical settings in which renal function is impaired, but its prevalence in the nephrotic syndrome has not been investigated in detail, even though this syndrome provides an excellent model in which to study a possible link between albuminuria, proteinuria, and hyperhomocyst(e)inemia. We obtained plasma and urine from 27 patients with biopsy-confirmed membranous glomerulonephritis presenting nephrotic syndrome and 27 matched controls and determined the concentrations of homocyst(e)ine and proteins considered putative markers of glomerular and tubular function. Hyperhomocyst(e)inemia, defined as the mean +SD of the plasma homocyst(e)ine concentration of the controls [plasma homocyst(e)ine concentration >10.8 µmol/l] was present in 26% of the patients with nephrotic syndrome but in only 7.4% of the controls. Furthermore, the degree of hyperhomocyst(e)inemia was more severe in the nephrotic patients than in the controls. The existence of renal failure, tubular damage, and, interestingly, relatively well conserved glomerular function barrier were the main predictors of increased levels of plasma homocyst(e)ine. In conclusion, hyperhomocyst(e)inemia is a frequent cardiovascular risk factor present in patients with nephrotic syndrome and renal failure, but it is not directly associated with proteinuria.

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]

We studied the effect in vitro and in vivo of dipyrone on the determination of several biochemical tests in two analyzers, a Hitachi 747 and a Kodak Ektachem 700. From studies in vitro, we found significant interference by dipyrone (P < 0.05) in the determination of creatine kinase (CK), lactate dehydrogenase (LD), uric acid, triglycerides, cholesterol, aspartate aminotransferase, alanine aminotransferase, and urea nitrogen with both instruments, and in the determination of creatinine in the Ektachem analyzer. We also studied the effect of intravenously administered dipyrone in 14 patients. Dipyrone interfered significantly (P < 0.05) in the determination of CK, LD, uric acid, triglycerides, and cholesterol with both instruments, and creatinine only with the Ektachem analyzer. Using high-performance liquid chromatography (HPLC), we measured concentrations of dipyrone in the serum of patients who had received the drug and observed a negative correlation between the concentrations of dipyrone in the blood and the percentage of each analyte concentration.