The oxidative modification of LDL appears to be involved in the development of various degenerative diseases such as atherosclerosis, carcinogenesis, aging and diabetes mellitus [11
]. Standard reference methods to prepare LDL from plasma employ ultracentrifugation. However, the selective precipitation methods, which are more accessible than ultracentrifugation, are widely used in the clinical laboratory for the measurement of the cholesterol content in different lipoprotein fractions. In particular, selective precipitation of LDL may be approached in different ways: by addition of heparin at an exactly controlled pH of 5.12 in the absence of divalent cations; or with polyvinylsulphate in the presence of EDTA and polyethylene glycol methyl ether; with amphipathic polymers in imidazole buffer at pH 6.10 (bioMerieux). An excellent statistical correlation is obtained when these methods are compared with reference ultracentrifugation methods, providing samples with triglyceride concentration above 8 mmol/l and those from patients with hyperlipoproteinemia Type III are excluded [13
]. In particular, the precipitating reagent used in the present work (bioMerieux), shows a good correlation coefficient (r = 0.96) when compared with ultracentrifugation methods [3
]. Its selectivity and the preservation of the immunological properties as well as the lipid composition of the native original LDLs have also been demonstrated [3
]. In our present study, we were unable to find differences in agarose electrophoretic mobility between LDL fractions obtained by this method of selective precipitation and ultracentrifugation. In addition, no contaminating lipoprotein fractions were observed by this electrophoretic method. In our standard procedure we washed the LDL precipitate once prior to solubilizing. Thus, it was necessary to establish whether there were changes in LDL cholesterol content, which could invalidate the original method's correlation with ultracentrifugation. However, we were unable to find cholesterol losses as a consequence of one or two washes with precipitating reagent.
Arshad et al. [16
] developed a simple method to assess whole plasma susceptibility to peroxidation by Cu2+
incubation. They used thiobarbituric acid reactivity to evaluate lipid peroxidation, a method which is not entirely specific. However, it proved to be easy to perform and accessible for the analysis of many samples. In the present work, we measured LDL-associated TBARS after induction of lipid peroxidation with a mixture of Cu2+
Several methodological aspects of our procedure were subsequently addressed, in order to achieve its optimization. a) The intra-assay precision was found to depend on the volume of solubilizing solution employed. In our standard working conditions, the CV was 4.8 %, which is lower than the precision limit established for the determination of selectively precipitated lipoprotein cholesterol (CV < 5%) [3
], and so can be considered acceptable. b) The observed percentage of recovery for exogenously added MDA (Table ) was comparable to that of the TBARS reaction (82-100 %) [10
]. These results suggest that the additioned MDA was still TBA reactive and did not generate any interfering substances, since the observed increment in MDA content did not significantly differ from that of the true value. c) It is important to ensure that the precipitate is not contaminated with non-LDL serum proteins, since results are expressed per LDL protein content. This contribution to variability was eliminated by washing the LDL precipitate. d) When the composition of the solubilizing solution was evaluated, precipitate redissolution effectively occurred in 50 g/l NaCl. However, the addition of Triton X-100 was chosen because it shortened the period of LDL redissolution. e) Lipid peroxidation kinetics have been extensively studied [8
]. It is known that LDL oxidation in the presence of Cu2+
shows three phases: latency, propagation and decomposition. This has been established by determination of hydroperoxides, TBARS or other aldehydes, fluorescent products and conjugated dienes. It has been shown that during the latency and propagation phases, as well as during the early stages of the decomposition phase, the time-courses of diene, TBARS and lipid hydroperoxide formation, are practically coincident [8
]. Indeed, the corresponding maxima coincide temporally. However, each individual's LDL shows its own particular kinetics so that sample to sample variations could represent a problem when - as in the present study - a single measurement of only one parameter is taken after a long incubation time. This does not allow us to conclusively establish whether the sample is at the end of its propagation phase, or has already begun its decomposition phase. In our preliminary studies of TBARS time-course, we found a lag phase followed by a maximum slope which ended at 150 min, the time point adopted for our standard procedure. A slower increment in absorbance was observed from this point on, a fact that may have been due to the decomposition of accumulated products. f) In the absence of oxidation inhibitors, LDL oxidation may continue throughout the TBA reaction period, thus contributing to the method's variability. This was effectively prevented by the addition of EDTA prior to the TBA reaction, which acts as an inhibitor of LDL oxidation by Cu2+
sequestration. g) Our experiments show that the combination of Cu2+
is more effective for the induction of LDL oxidation, than each agent its own. The observed results suggest a synergistic mechanism of action between both reagents. Previous studies have addressed the Cu2+
-induced in vitro oxidation of plasma LDL [17
]. These authors found a value of 21 ± 3 nmol MDA / mg LDL protein, obtained from four normal subjects, for LDL isolated by ultracentrifugation. This is practically coincident with the results which we obtained with our control healthy population (21.7 ± 1.5 nmol MDA / mg LDL protein), as would be expected from the reported correlation between LDL obtained by ultracentrifugation and by the LDL-precipitating method of bioMerieux. Recently, Guerci et al.[14
] studied the LDL oxidation susceptibility of normolipidemic diabetic and non-diabetic patients. These authors found a significant increase in type 2 diabetic patients vs. healthy subjects, particularly in the group of type 2 diabetic females, in which LDL oxidation susceptibility was highest. In coincidence with these reported results, LDL oxidative susceptibility of our type 2 diabetic patients was significantly greater (39.0 ± 3.0 nmol MDA / mg LDL protein) than the control group.
The LDL precipitation method which we have used in this study is based on interaction with glycosaminoglycans (GAG). However, both lipid composition and the content of sialic acid can modulate the interaction with GAG. In this context, particles such as small dense LDL can interact with GAG with high affinity. In addition, the precipitation procedure may increase the susceptibility for oxidation by copper since copper penetrates the LDL particle more easily after precipitation. In consequence, we cannot discard the possibility that our results may reflect a preselection of LDL with higher susceptibility for oxidation.